Methods and compositions for enhancing an immune response, blocking monocyte migration, amplifying vaccine immunity and inhibiting tumor growth and metastasis

ABSTRACT

Provided are methods of enhancing an immune response and methods for reducing the recruitment of monocytes to a lymph node by administering to an individual an angiotensin II receptor blocker or a compound of Formula (I) in conjunction with an antigen. The invention also provides relate methods for amplifying vaccine immunity by administering to an individual an angiotensin II receptor blocker or a compound of Formula (I) in conjunction with an vaccine. The invention also provides related methods of inhibiting tumor growth and metastasis by administering to an individual with cancer an angiotensin II receptor blocker or a compound of Formula (I) in conjunction with an anti-tumor preparation. In addition, related compositions comprising an ARB or a compound of Formula (I) and an antigen, vaccine, or anti-tumor preparation are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/195,688, filed Mar. 3, 2014, which claims priority to U.S.Provisional Application No. 61/771,738, filed on Mar. 1, 2013, and U.S.Provisional Application No. 61/771,744, filed on Mar. 1, 2013, each ofwhich is incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention provides methods for enhancing an immune response,decreasing monocyte recruitment to lymph nodes, amplifying vaccineimmunity, and reducing tumor growth or metastasis. The invention alsoincludes compositions and kits used to practice these methods.

BACKGROUND OF THE INVENTION

Vaccine adjuvant-induced inflammation augments vaccine immunity in partby recruiting antigen presenting myeloid cells (monocytes andneutrophils) to vaccine draining lymph nodes (LNs) (Serafini et al.(2004) Cancer Res. 64:6337-6343; Martino et al. (2010) J. Immunol. 184:2038-2047). However, recent evidence indicates that monocyte recruitmentto LNs suppresses local B cell and T cell activation and proliferation(Mitchell et al. (2012) J. Immunology 189: 5612-5621; Mitchell et al.(2012) Int. Immunopharmacol. 15: 357-363). Lowered immune responsesfollowing vaccination can lead to decreased vaccine efficacy.

Moreover, the role of immature and immune suppressive myeloid cells,including neutrophils, monocytes, and tumor-associated macrophages, inpromoting the growth of primary tumors is well established¹⁻¹⁰.Additionally, myeloid cells, and especially monocytes play an importantrole in creating favorable conditions for the seeding and growth oftumor metastases in the lungs, in part by establishing the so-calledmetastatic niche¹⁵⁻¹⁷. Inflammatory monocytes recruited in response totumor-derived signals have been shown to play a key role in promotingthe growth of tumor metastases. The major chemokine regulating monocyterecruitment is MCP-1 (CCL2), which signals primarily via activation ofthe receptor CCR2 expressed principally on inflammatory monocytes.

What are needed in the art, are methods for administering vaccines andrelated vaccine compositions that enhance the immune response to anantigen and augment vaccine efficacy by inhibiting the suppressiveeffects of monocytes at LNs and enhancing B cell and T cell responses.What is also needed in the art, are methods for inhibiting the migrationof myeloid cells, and in particular inflammatory monocytes, to the siteof tumors where they act to promote tumor growth and metastasis. Thepresent invention is directed to overcoming these deficiencies in theart.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the invention provides a method of enhancing an immuneresponse against an antigen in an individual, the method comprising:

-   -   (a) administering to the individual an effective amount of the        antigen in conjunction with a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof; or    -   (b) administering to the individual an effective amount of an        angiotensin II receptor blocker (ARB) in conjunction with the        antigen, thereby enhancing the immune response.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II), Formula (III), or Formula (IV), as described herein. Incertain embodiments, the compound of Formula (I) is Ondansteron orAlosetron. In certain embodiments, the ARB is Losartan, Candesartan,Eprosartan, Irbesartan, Olmesartan, Telmisartan, Azilsartan, orValsartan.

In certain embodiments, the enhanced immune response comprises anenhanced humoral immune response. In certain embodiments, the enhancedhumoral immune response comprises an increased antibody titer againstthe antigen. In certain embodiments, the enhanced immune responsecomprises an enhanced cellular immune response. In certain embodiments,the enhanced cellular immune response comprises increased release ofIFNγ in response to the antigen. In certain embodiments, the enhancedimmune response comprises an enhanced humoral immune response and anenhanced cellular immune response.

In certain embodiments, the antigen comprises live whole virus, killedwhole virus, attenuated whole virus, killed bacteria, attenuatedbacteria, a virus-like particle, a bacterial, viral, or parasiteprotein, a recombinant protein, or a peptide.

In certain embodiments comprising administering an ARB, the individualreceiving an ARB does not have hypertension, congestive heart failure, ahistory of myocardial infarction, or diabetic nephropathy. In certainembodiments, the individual receiving an ARB has not taken the ARB forthe treatment of hypertension, congestive heart failure, or diabeticnephropathy. In certain embodiments, the individual receiving an ARBdoes not have a detectable level of the ARB in their blood or urineprior to administration of the ARB in conjunction with the antigen.

In certain embodiments, the antigen and the ARB are present in a singlepharmaceutical composition. In certain embodiments, the antigen and thecompound of Formula (I) are present in a single pharmaceuticalcomposition. In certain embodiments, the single pharmaceuticalcomposition is administered orally, via topical application, inhalation,intravenous injection, intra-arterial injection, intramuscularinjection, application to a wound site, application to a surgical site,intracavitary injection, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intraplurally, intraperitoneally,intraventricularly, intra-articularly, intraocularly, or intraspinally.

In certain embodiments, the ARB is present in a first pharmaceuticalcomposition, and the antigen is present in a second pharmaceuticalcomposition. In certain embodiments, the compound of Formula (I) ispresent in a first pharmaceutical composition, and the antigen ispresent in a second pharmaceutical composition. In certain embodiments,the first pharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally. In certain embodiments, the secondpharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally. In certain embodiments, the firstpharmaceutical composition is administered before the secondpharmaceutical composition. In certain embodiments, the firstpharmaceutical composition is administered after the secondpharmaceutical composition. In certain embodiments, the first and secondpharmaceutical compositions are administered within a time period ofless than 12 hours of one another. In certain embodiments, the firstpharmaceutical composition and the second pharmaceutical compositionsare administered simultaneously.

In certain embodiments, the ARB is Losartan, and the Losartan isadministered at a dosage of 30 mg/kg. In certain embodiments, the ARB isLosartan, and the Losartan is administered at a dosage of less than 25mg. In certain embodiments, the ARB is Candesartan, and the Candesartanis administered at a dosage of less than 4 mg. In certain embodiments,the ARB is Eprosartan, and the Eprosartan is administered at a dosage ofless than 400 mg. In certain embodiments, the ARB is Irbesartan, and theIrbesartan is administered at a dosage of less than 150 mg. In certainembodiments, the ARB is Olmesartan, and the Olmesartan is administeredat a dosage of less than 20 mg. In certain embodiments, the ARB isTelmisartan, and the Telmisartan is administered at a dosage of lessthan 20 mg. In certain embodiments, the ARB is Valsartan, and theValsartan is administered at a dosage of less than 20 mg. In certainembodiments, the ARB is Azilsartan, and the Azilsartan is administeredat a dosage of less than 80 mg.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (III):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IV):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (V):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is Ondansetron. In someembodiments, the Ondansetron is administered at a dosage of 3 mg/kg. Insome embodiments, the Ondansetron is administered at a dosage of lessthan 12 mg. In some embodiments, the compound of Formula (I) isAlosetron. In some embodiments, the Alosetron is administered at adosage of less than 0.5 mg.

In some embodiments comprising administering a Ondansetron or Alosetron,the individual has not taken the Ondansetron or the Alosetron for thetreatment of irritable bowel syndrome (IBS), post-operative nausea andvomiting (PONV), radiation-induced nausea and vomiting (RINV), orchemotherapy-induced nausea and vomiting (CINV). In some embodiments,the individual does not have a detectable level of the Ondansetron orthe Alosetron in their blood or urine prior to administration of theOndansetron or the Alosetron in conjunction with the antigen

In some embodiments, the present invention provides a method ofdecreasing recruitment of monocytes to a lymph node in an individual,the method comprising:

-   -   (a) administering to the individual an antigen in conjunction        with an effective amount of a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof; or    -   (b) administering to the individual an effective amount of an        angiotensin II receptor blocker (ARB) in conjunction with an        antigen, thereby decreasing the recruitment of the monocytes to        lymph node.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II), Formula (III), or Formula (IV), as described herein. Incertain embodiments, the compound of Formula (I) is Ondansteron orAlosetron.

In certain embodiments, the ARB is Losartan, Candesartan, Eprosartan,Irbesartan, Olmesartan, Telmisartan, Azilsartan, or Valsartan.

In certain embodiments, the monocytes are inflammatory monocytes orCD14^(hi)CD16⁻ human monocytes. In certain embodiments, the lymph nodeis a draining lymph node. In certain embodiments, the draining lymphnode is a vaccine draining lymph node.

In certain embodiments comprising administering an ARB, the enhancedimmune response comprises an enhanced humoral immune response. Incertain embodiments, the enhanced humoral immune response comprises anincreased antibody titer against the antigen. In certain embodiments,the enhanced immune response comprises an enhanced cellular immuneresponse. In certain embodiments, the enhanced cellular immune responsecomprises increased release of IFNγ in response to the antigen. Incertain embodiments, the enhanced immune response comprises an enhancedhumoral immune response and an enhanced cellular immune response.

In certain embodiments, the antigen comprises live whole virus, killedwhole virus, attenuated whole virus, killed bacteria, attenuatedbacteria, a virus-like particle, a bacterial, viral, or parasiteprotein, a recombinant protein, or a peptide.

In certain embodiments comprising administering an ARB, the individualreceiving an ARB does not have hypertension, congestive heart failure, ahistory of myocardial infarction, or diabetic nephropathy. In certainembodiments, the individual receiving an ARB has not taken the ARB forthe treatment of hypertension, congestive heart failure, or diabeticnephropathy. In certain embodiments, the individual receiving an ARBdoes not have a detectable level of the ARB in their blood or urineprior to administration of the ARB in conjunction with the antigen.

In certain embodiments, the antigen and the ARB are present in a singlepharmaceutical composition. In certain embodiments, the antigen and thecompound of Formula (I) are present in a single pharmaceuticalcomposition. In certain embodiments, the single pharmaceuticalcomposition is administered orally, via topical application, inhalation,intravenous injection, intra-arterial injection, intramuscularinjection, application to a wound site, application to a surgical site,intracavitary injection, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intraplurally, intraperitoneally,intraventricularly, intra-articularly, intraocularly, or intraspinally.

In certain embodiments, the ARB is present in a first pharmaceuticalcomposition and the antigen is present in a second pharmaceuticalcomposition. In certain embodiments, the compound of Formula (I) ispresent in a first pharmaceutical composition and the antigen is presentin a second pharmaceutical composition. In certain embodiments, thefirst pharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally. In certain embodiments, the secondpharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally. In certain embodiments, the firstpharmaceutical composition is administered before the secondpharmaceutical composition. In certain embodiments, the firstpharmaceutical composition is administered after the secondpharmaceutical composition. In certain embodiments, the first and secondpharmaceutical compositions are administered within a time period ofless than 12 hours of one another. In certain embodiments, the firstpharmaceutical composition and the second pharmaceutical compositionsare administered simultaneously.

In certain embodiments, the ARB is Losartan, and the Losartan isadministered at a dosage of 30 mg/kg. In certain embodiments, the ARB isLosartan, and the Losartan is administered at a dosage of less than 25mg. In certain embodiments, the ARB is Candesartan, and the Candesartanis administered at a dosage of less than 4 mg. In certain embodiments,the ARB is Eprosartan, and the Eprosartan is administered at a dosage ofless than 400 mg. In certain embodiments, the ARB is Irbesartan, and theIrbesartan is administered at a dosage of less than 150 mg. In certainembodiments, the ARB is Olmesartan, and the Olmesartan is administeredat a dosage of less than 20 mg. In certain embodiments, the ARB isTelmisartan, and the Telmisartan is administered at a dosage of lessthan 20 mg. In certain embodiments, the ARB is Valsartan, and theValsartan is administered at a dosage of less than 20 mg. In certainembodiments, the ARB is Azilsartan, and the Azilsartan is administeredat a dosage of less than 80 mg.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (III):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is s a compound ofFormula (IV):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (V):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is Ondansetron. In someembodiments, the Ondansetron is administered at a dosage of 3 mg/kg. Insome embodiments, the Ondansetron is administered at a dosage of lessthan 12 mg. In some embodiments, the compound of Formula (I) isAlosetron. In some embodiments, the Alosetron is administered at adosage of less than 0.5 mg.

In some embodiments, comprising administering a Ondansetron orAlosetron, the individual has not taken the Ondansetron or the Alosetronfor the treatment of irritable bowel syndrome (IBS), post-operativenausea and vomiting (PONV), radiation-induced nausea and vomiting(RINV), or chemotherapy-induced nausea and vomiting (CINV). In someembodiments, the individual does not have a detectable level of theOndansetron or the Alosetron in their blood or urine prior toadministration of the Ondansetron or the Alosetron in conjunction withthe antigen

In some embodiments, the present invention provides a method ofamplifying vaccine immunity in an individual, the method comprising

-   -   (a) administering to the individual the vaccine in conjunction        with an effective amount of an ARB or a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof; or    -   (b) administering to the individual the vaccine in conjunction        with an effective amount of an angiotensin receptor blocker        (ARB), thereby amplifying the immunity of the vaccine in the        individual.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II), Formula (III), or Formula (IV), as described herein. Incertain embodiments, the compound of Formula (I) is Ondansteron orAlosetron. In certain embodiments, the ARB is Losartan, Candesartan,Eprosartan, Irbesartan, Olmesartan, Telmisartan, Azilsartan, orValsartan.

In certain embodiments, the vaccine comprises live whole virus, killedwhole virus, attenuated whole virus, killed bacteria, attenuatedbacteria, a virus-like particle, a bacterial, viral, or parasiteprotein, a recombinant protein, or a peptide.

In certain embodiments comprising administering an ARB, the individualreceiving an ARB does not have hypertension, congestive heart failure, ahistory of myocardial infarction, or diabetic nephropathy. In certainembodiments, the individual receiving an ARB has not taken the ARB forthe treatment of hypertension, congestive heart failure, or diabeticnephropathy. In certain embodiments, the individual receiving an ARBdoes not have a detectable level of the ARB in their blood or urineprior to administration of the ARB in conjunction with the antigen orvaccine.

In certain embodiments, the antigen or vaccine and the ARB are presentin a single pharmaceutical composition. In certain embodiments, thevaccine and the compound of Formula (I) are present in a singlepharmaceutical composition. In certain embodiments, the singlepharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally.

In certain embodiments, the ARB is present in a first pharmaceuticalcomposition and the antigen or vaccine is present in a secondpharmaceutical composition. In certain embodiments, the compound ofFormula (I) is present in a first pharmaceutical composition and thevaccine is present in a second pharmaceutical composition. In certainembodiments, the first pharmaceutical composition is administeredorally, via topical application, inhalation, intravenous injection,intra-arterial injection, intramuscular injection, application to awound site, application to a surgical site, intracavitary injection, bysuppository, subcutaneously, intradermally, transcutaneously, bynebulization, intraplurally, intraperitoneally, intraventricularly,intra-articularly, intraocularly, or intraspinally. In certainembodiments, the second pharmaceutical composition is administeredorally, via topical application, inhalation, intravenous injection,intra-arterial injection, intramuscular injection, application to awound site, application to a surgical site, intracavitary injection, bysuppository, subcutaneously, intradermally, transcutaneously, bynebulization, intraplurally, intraperitoneally, intraventricularly,intra-articularly, intraocularly, or intraspinally. In certainembodiments, the first pharmaceutical composition is administered beforethe second pharmaceutical composition. In certain embodiments, the firstpharmaceutical composition is administered after the secondpharmaceutical composition. In certain embodiments, the first and secondpharmaceutical compositions are administered within a time period ofless than 12 hours of one another. In certain embodiments, the firstpharmaceutical composition and the second pharmaceutical compositionsare administered simultaneously.

In certain embodiments, the ARB is Losartan, and the Losartan isadministered at a dosage of 30 mg/kg. In certain embodiments, the ARB isLosartan, and the Losartan is administered at a dosage of less than 25mg. In certain embodiments, the ARB is Candesartan, and the Candesartanis administered at a dosage of less than 4 mg. In certain embodiments,the ARB is Eprosartan, and the Eprosartan is administered at a dosage ofless than 400 mg. In certain embodiments, the ARB is Irbesartan, and theIrbesartan is administered at a dosage of less than 150 mg. In certainembodiments, the ARB is Olmesartan, and the Olmesartan is administeredat a dosage of less than 20 mg. In certain embodiments, the ARB isTelmisartan, and the Telmisartan is administered at a dosage of lessthan 20 mg. In certain embodiments, the ARB is Valsartan, and theValsartan is administered at a dosage of less than 20 mg. In certainembodiments, the ARB is Azilsartan, and the Azilsartan is administeredat a dosage of less than 80 mg.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;        is a single bond or double bond;    -   Q¹ is N or CH;    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (III):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IV):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof

In some embodiments, the compound of Formula (I) is a compound ofFormula (V):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is Ondansetron. In someembodiments, the Ondansetron is administered at a dosage of 3 mg/kg. Insome embodiments, the Ondansetron is administered at a dosage of lessthan 12 mg. In some embodiments, the compound of Formula (I) isAlosetron. In some embodiments, the Alosetron is administered at adosage of less than 0.5 mg.

In some embodiments comprising administering a Ondansetron or Alosetron,the individual has not taken the Ondansetron or the Alosetron for thetreatment of irritable bowel syndrome (IBS), post-operative nausea andvomiting (PONV), radiation-induced nausea and vomiting (RINV), orchemotherapy-induced nausea and vomiting (CINV). In some embodiments,the individual does not have a detectable level of the Ondansetron orthe Alosetron in their blood or urine prior to administration of theOndansetron or the Alosetron in conjunction with the antigen or vaccine.

In some embodiments, the present invention provides a method ofinhibiting tumor growth or metastasis in an individual with cancer, themethod comprising

-   -   (a) administering to the individual an anti-tumor preparation in        conjunction with an effective amount of a compound of Formula        (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;    -   or pharmaceutically acceptable salts thereof; or    -   (b) administering to the individual an anti-tumor preparation in        conjunction with an effective amount of an angiotensin II        receptor blocker (ARB), thereby inhibiting tumor growth or        metastasis in the individual.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II), Formula (III), or Formula (IV), as described herein. Incertain embodiments, the compound of Formula (I) is Ondansteron orAlosetron. In certain embodiments, the ARB is Losartan, Candesartan,Eprosartan, Irbesartan, Olmesartan, Telmisartan, Azilsartan, orValsartan.

In certain embodiments, the anti-tumor preparation comprises atherapeutic antibody, a topoisomerase inhibitor, an antimetabolite, aplatinum-based agent, an alkylating agent, a tyrosine kinase inhibitor,an Anthracycline antibiotic, an anti-angiogenic agent, or a vincaalkaloid. In certain embodiments, the tyrosine kinase inhibitor isSunitinib.

In certain embodiments, the cancer is an epithelial cancer, breastcancer, prostate cancer, colon cancer, a hematopoietic cancer, leukemia,lymphoma, a sarcoma, melanoma, a head sarcoma, a neck sarcoma, asquamous cell carcinoma, an osteosarcoma, or a brain tumor.

In certain embodiments comprising administering an ARB, the individualreceiving an ARB does not have hypertension, congestive heart failure, ahistory of myocardial infarction, or diabetic nephropathy. In certainembodiments, the individual receiving an ARB has not taken the ARB forthe treatment of hypertension, congestive heart failure, or diabeticnephropathy. In certain embodiments, the individual receiving an ARBdoes not have a detectable level of the ARB in their blood or urineprior to administration of the ARB in conjunction with the antigen oranti-tumor preparation.

In certain embodiments, the anti-tumor preparation and the ARB arepresent in a single pharmaceutical composition. In certain embodiments,the anti-tumor preparation and the compound of Formula (I) are presentin a single pharmaceutical composition. In certain embodiments, thesingle pharmaceutical composition is administered orally, via topicalapplication, inhalation, intravenous injection, intra-arterialinjection, intramuscular injection, application to a wound site,application to a surgical site, intracavitary injection, by suppository,subcutaneously, intradermally, transcutaneously, by nebulization,intraplurally, intraperitoneally, intraventricularly, intra-articularly,intraocularly, or intraspinally.

In certain embodiments, the ARB is present in a first pharmaceuticalcomposition, and the anti-tumor preparation is present in a secondpharmaceutical composition. In certain embodiments, the compound ofFormula (I) is present in a first pharmaceutical composition, and theanti-tumor preparation is present in a second pharmaceuticalcomposition. In certain embodiments, the first pharmaceuticalcomposition is administered orally, via topical application, inhalation,intravenous injection, intra-arterial injection, intramuscularinjection, application to a wound site, application to a surgical site,intracavitary injection, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intraplurally, intraperitoneally,intraventricularly, intra-articularly, intraocularly, or intraspinally.In certain embodiments, the second pharmaceutical composition isadministered orally, via topical application, inhalation, intravenousinjection, intra-arterial injection, intramuscular injection,application to a wound site, application to a surgical site,intracavitary injection, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intraplurally, intraperitoneally,intraventricularly, intra-articularly, intraocularly, or intraspinally.In certain embodiments, the first pharmaceutical composition isadministered before the second pharmaceutical composition. In certainembodiments, the first pharmaceutical composition is administered afterthe second pharmaceutical composition. In certain embodiments, the firstand second pharmaceutical compositions are administered within a timeperiod of less than 12 hours of one another. In certain embodiments, thefirst pharmaceutical composition and the second pharmaceuticalcompositions are administered simultaneously.

In certain embodiments, the ARB is Losartan, and the Losartan isadministered at a dosage of 30 mg/kg. In certain embodiments, the ARB isLosartan, and the Losartan is administered at a dosage of less than 25mg. In certain embodiments, the ARB is Candesartan, and the Candesartanis administered at a dosage of less than 4 mg. In certain embodiments,the ARB is Eprosartan, and the Eprosartan is administered at a dosage ofless than 400 mg. In certain embodiments, the ARB is Irbesartan, and theIrbesartan is administered at a dosage of less than 150 mg. In certainembodiments, the ARB is Olmesartan, and the Olmesartan is administeredat a dosage of less than 20 mg. In certain embodiments, the ARB isTelmisartan, and the Telmisartan is administered at a dosage of lessthan 20 mg. In certain embodiments, the ARB is Valsartan, and theValsartan is administered at a dosage of less than 20 mg. In certainembodiments, the ARB is Azilsartan, and the Azilsartan is administeredat a dosage of less than 80 mg.

In some embodiments, the ARB is losartan, and the anti-tumor preparationis a tyrosine kinase inhibitor. In one certain embodiment, the ARB islosartan and the anti-tumor preparation is the tyrosine kinase inhibitorsunitinib.

In some embodiments, the compound of Formula (I) is a compound ofFormula (II):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (III):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (IV):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is a compound ofFormula (V):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments, the compound of Formula (I) is Ondansetron. In someembodiments, the Ondansetron is administered at a dosage of 3 mg/kg. Insome embodiments, the Ondansetron is administered at a dosage of lessthan 12 mg. In some embodiments, the compound of Formula (I) isAlosetron. In some embodiments, the Alosetron is administered at adosage of less than 0.5 mg.

In some embodiments, comprising administering Ondansetron or Alosetron,the individual has not taken the Ondansetron or the Alosetron for thetreatment of irritable bowel syndrome (IBS), post-operative nausea andvomiting (PONV), radiation-induced nausea and vomiting (RINV), orchemotherapy-induced nausea and vomiting (CINV). In some embodiments,the individual does not have a detectable level of the Ondansetron orthe Alosetron in their blood or urine prior to administration of theOndansetron or the Alosetron in conjunction with the antigen oranti-tumor preparation.

In some embodiments, the present invention provides a compositioncomprising (i) an angiotensin II receptor blocker (ARB) and (ii) anantigen or a vaccine. In certain embodiments, the ARB is Azilsartan,Losartan, Candesartan, Eprosartan, Irbesartan, Olmesartan, Telmisartan,or Valsartan. Also provided by the invention is a (pharmaceutical)composition comprising (i) an angiotensin II receptor blocker (ARB),(ii) an antigen or a vaccine, and (iii) a pharmaceutically acceptablecarrier. In certain embodiments, the antigen comprises live whole virus,killed whole virus, attenuated whole virus, killed bacteria, attenuatedbacteria, a virus-like particle, a bacterial, viral, or parasiteprotein, a recombinant protein, or a peptide.

In some embodiments, the invention also provides compositions comprising(i) an antigen or a vaccine, and (ii) a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the composition further comprises (iii) apharmaceutically acceptable carrier. In some embodiments, the compoundof Formula (I) is a compound of Formula (II), Formula (III), or Formula(IV), as described herein. In some embodiments, the compound of Formula(I) is Alosetron or Ondansetron. In some embodiments, the antigen or thevaccine comprises live whole virus, killed whole virus, attenuated wholevirus, killed bacteria, attenuated bacteria, a virus-like particle, abacterial, viral, or parasite protein, a recombinant protein, or apeptide.

In some embodiments, the invention provides a composition comprising (i)an anti-tumor preparation and (ii) an angiotensin II receptor blocker(ARB). In certain embodiments, the ARB is Azilsartan, Losartan,Candesartan, Eprosartan, Irbesartan, Olmesartan, Telmisartan, orValsartan. The invention also provides pharmaceutical compositionscomprising (i) an anti-tumor preparation, (ii) an angiotensin IIreceptor blocker (ARB), and (iii) a pharmaceutically acceptable carrier.In certain embodiments, the anti-tumor preparation comprises atherapeutic antibody, a topoisomerase inhibitor, an antimetabolite, aplatinum-based agent, an alkylating agent, a tyrosine kinase inhibitor,an Anthracycline antibiotic, an anti-angiogenic agent, or a vincaalkaloid. In certain embodiments, the anti-tumor preparation comprisesthe tyrosine kinase inhibitor sunitinib.

In some embodiments, the invention provides compositions comprising (i)an anti-tumor preparation and (ii) a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments, the composition further comprises (iii) apharmaceutically acceptable carrier. In certain embodiments, theanti-tumor preparation comprises a therapeutic antibody, a topoisomeraseinhibitor, an antimetabolite, a platinum-based agent, an alkylatingagent, a tyrosine kinase inhibitor, an Anthracycline antibiotic, ananti-angiogenic agent, or a vinca alkaloid. In certain embodiments thecompound of Formula (I) is Alosetron or Ondansetron. In certainembodiments, the tyrosine kinase inhibitor is sunitinib.

The invention also provides kits comprising a composition describedherein and instructions for use.

The invention also provides kits comprising (i) a vaccine, an antigen,or an anti-tumor preparation, and (ii) a compound of Formula (I)

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof, wherein (i) the        compound of Formula (I) is provided in a first container, and        wherein (ii) the vaccine, antigen, or anti-tumor preparation is        provided in a second container. In some embodiments, the        compound of Formula (I) is a compound of Formula (II), Formula        (III), or Formula (IV), as described herein. In some        embodiments, the compound of Formula (I) is Alosetron or        Ondansetron.

In certain embodiments, the kit optionally further comprises (iii)instructions for use.

In certain embodiments, kits of the invention comprise (i) an ARB and(ii) a vaccine, an antigen, or an anti-tumor preparation, wherein theARB is provided in a first container, and wherein the vaccine, antigen,or anti-tumor preparation is provided in a second container. In someembodiments of the kits, the ARB is Azilsartan, Losartan, Candesartan,Eprosartan, Irbesartan, Olmesartan, Telmisartan, or Valsartan.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of experiments conducted to assess the abilityof Losartan to inhibit human monocyte migration in vitro.

FIG. 2 shows the results of experiments conducted to assess the abilityof Losartan to inhibit canine monocyte migration in vitro.

FIG. 3 shows the results of experiments conducted to assess the abilityof Ondansetron to inhibit human monocyte migration in vitro.

FIG. 4 shows the results of experiments conducted to determine theeffect of Ondansetron treatment on the migration of immune suppressiveinflammatory monocytes to lymph nodes following vaccination in mice.

FIG. 5 provides a quantitative analysis of the results shown in FIG. 4.

FIG. 6 shows the results of experiments conducted to determine theeffect of Losartan treatment on the migration of immune suppressiveinflammatory monocytes to lymph nodes following vaccination in mice.

FIGS. 7a and 7b show the results of experiments conducted to determinethe effect of Losartan treatment on the migration of circulating immunesuppressive inflammatory monocytes in healthy dogs. (a) and (b) show theresults from two different dogs.

FIG. 8 shows the results of experiments conducted to determine theeffect of Losartan treatment on the migration of circulating immunesuppressive inflammatory monocytes in dogs with cancer.

FIG. 9 shows the results of experiments conducted to determine theeffect of Losartan treatment on a humoral immune response.

FIG. 10 shows the results of experiments conducted to determine theeffect of Ondansetron treatment on a humoral immune response.

FIG. 11 shows the results of experiments conducted to determine theeffect of Losartan treatment on a cellular immune response.

FIG. 12 shows the results of experiments conducted to determine theeffect of Ondansetron on tumor vaccine responses.

FIG. 13 shows the results of experiments conducted to determine theeffect of Ondansetron treatment on myeloid cell accumulation in tumortissues following vaccination.

FIG. 14 shows the results of an experiment conducted to determine thesynergistic interactions between losartan and sunitinib for inhibitingtumor growth.

FIGS. 15a and 15b show the results of preliminary experiments conductedto determine the synergistic interactions between losartan and sunitinibfor inhibiting monocyte migration. (a) shows the percentage ofCD11b+Ly6C+ monocytes present in the lungs of the mice having lungmetastases. (b) shows the frequency of PDL1+ monocytes to total cellspresent in the lungs of the mice having lung metastases.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides, inter alia, compositions, methods, and kits forenhancing an immune response in an individual, decreasing therecruitment of monocytes to a lymph node in an individual, amplifyingvaccine immunity in an individual, and reducing tumor growth ormetastasis in an individual with cancer.

Certain embodiments are based in part on the observation that compoundsof Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof, which are        typically indicated for treating nausea and vomiting, certain        forms of psychosis, symptoms of severe irritable bowel syndrome,        tardive dyskinesia, and forms of gastroenteritis, inhibit the        migration of monocytes (e.g., migration to vaccine draining        lymph nodes), inhibit the suppressive effects of monocytes on        vaccine immunity, and inhibit tumor growth and metastasis.

Certain embodiments of the invention are based in part on theobservation that angiotensin II receptor blockers (ARBs), which aretypically indicated for the treatment of hypertension, inhibit themigration of monocytes (e.g., migration to vaccine draining lymphnodes), inhibit the suppressive effects of monocytes on vaccineimmunity, and inhibit tumor growth and metastasis.

General Methods

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell culturing, molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry and immunology, which are within the skill of the art. Suchtechniques are explained fully in the literature, such as, MolecularCloning: A Laboratory Manual, third edition (Sambrook et al., 2001) ColdSpring Harbor Press; Oligonucleotide Synthesis (P. Herdewijn, ed.,2004); Animal Cell Culture (R. I. Freshney), ed., 1987); Methods inEnzymology (Academic Press, Inc.); Handbook of Experimental Immunology(D. M. Weir & C. C. Blackwell, eds.); Gene Transfer Vectors forMammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); CurrentProtocols in Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR:The Polymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); ShortProtocols in Molecular Biology (Wiley and Sons, 1999); Manual ofClinical Laboratory Immunology (B. Detrick, N. R. Rose, and J. D. Foldseds., 2006); Immunochemical Protocols (J. Pound, ed., 2003); Lab Manualin Biochemistry: Immunology and Biotechnology (A. Nigam and A. Ayyagari,eds. 2007); Immunology Methods Manual: The Comprehensive Sourcebook ofTechniques (Ivan Lefkovits, ed., 1996); Using Antibodies: A LaboratoryManual (E. Harlow and D. Lane, eds., 1988); and others.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs.

As used herein, the term “effective amount” refers to at least an amounteffective, at dosages and for periods of time necessary, to achieve thedesired result, e.g., an enhanced immune response to an antigen, adecrease in monocyte recruitment to a lymph node following vaccination,an amplification of vaccine immunity, an inhibition of tumor growth andmetastasis, etc. An effective amount can be provided in one or moreadministrations.

As used herein, the term “in conjunction with” refers to the temporalproperty of two events occurring at approximately the same time, e.g.,between 0-12 hours of one another. For example, when an ARB or acompound of Formula (I) is administered in conjunction with an antigenor a vaccine, the ARB or the compound of Formula (I) and the antigen orvaccine are administered within less than a 12 hour period. The term “inconjunction with” is not limited by the order in which the two eventsoccur.

As used herein, the term “individual” refers to a mammal, e.g., a human,a companion animal (e.g., dog, cat, rodent, rabbit, etc.), a sportanimal (e.g., horse, dog, bull, etc.), a farm or food animal (e.g., pig,cow, sheep, goat, etc.), livestock (e.g., donkeys, goats, guinea pigs,sheep, cattle, llamas, etc.), or any other mammalian veterinary animal.

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise.

Reference to “about” a value or parameter herein refers to the usualerror range for the respective value readily known to the skilled personin this technical field. Reference to “about” a value or parameterherein includes (and describes) aspects that are directed to that valueor parameter per se. For example, description referring to “about X”includes description of “X.”

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

Compositions

During vaccination, an antigen is administered to an individual tostimulate antigen-specific immunity, thus increasing the individual'sresistance (or decreasing his or her susceptibility) to a particulardisease or infection. In order for vaccination to be effective, theantigen must be capable of provoking robust humoral (i.e., B cell)and/or cell-mediated (i.e., T cell) immune responses. However, CCR2⁺inflammatory monocytes, which are recruited to the site of vaccinationand to the vaccine draining lymph nodes, have been shown to have asuppressive effect on B cell and T cell responses (Mitchell et al.(2012) J. Immunology 189: 5612-5621).

In certain embodiments, compositions that include both (i) anangiotensin II receptor blocker (ARB) or a compound of Formula (I); and(ii) an antigen, a vaccine, or an anti-tumor preparation, as describedin greater detail herein, are used to reduce monocyte recruitment tolymph nodes, enhance the individual's immune response against theantigen, amplify vaccine immunity, or inhibit tumor growth ormetastasis.

Similarly, in certain embodiments, a composition that includes anangiotensin II receptor blocker (ARB) or a compound of Formula (I), whenused together with a separate composition that includes an antigen, avaccine, or an anti-tumor preparation, as described in greater detailherein, are used to reduce monocyte recruitment to lymph nodes, enhancethe individual's immune response against the antigen, amplify vaccineimmunity, or inhibit tumor growth or metastasis.

Accordingly, in certain embodiments, the present invention provides acomposition comprising an ARB or a Compound of Formula (I). In certainembodiments, the present invention includes a composition comprising anantigen, a vaccine, or a tumor preparation. In certain embodiments, thepresent invention provides a composition comprising (i) an ARB and (ii)an antigen, a vaccine, or a tumor preparation. In certain embodiments,the present invention provides a composition comprising (i) a Compoundof Formula (I) and (ii) an antigen, a vaccine, or a tumor preparation.In certain embodiments, such compositions further comprise apharmaceutically acceptable carrier. In certain embodiments, suchcompositions are pharmaceutically acceptable compositions. In certainembodiments, such compositions are administered to an individual. Incertain embodiments, such compositions are used to reduce monocyterecruitment to lymph nodes, enhance the individual's immune responseagainst the antigen, amplify vaccine immunity, or to inhibit tumorgrowth or metastasis.

Angiotensin II Receptor Blockers (ARBs)

Certain embodiments of the invention relate to Angiotensin II receptorblockers or “ARBs” herein (also known as angiotensin receptorantagonists, AT₁-receptor antagonists, or sartans). ARBs are a group ofpharmaceuticals which modulate the renin-angiotensin-aldosterone systemby selectively inhibiting the effects of angiotensin II (Ang II), apeptide hormone that plays an important role in the pathophysiology ofhypertension. ARBs antagonize the action of Ang II at the Ang II type 1(AT₁) receptor and produce their blood pressure lowering effects byreversing the effects of Ang II, including, e.g., vasoconstriction,aldosterone release, ADH secretion, ACTH secretion, increased sodiumabsorption by the kidney, and catecholamine release. Losartan was thefirst ARB to be developed and approved by the United States Food andDrug Administration (FDA), and it has served as the basis for thedevelopment of other ARBs, including Azilsartan, Candesartan,Eprosartan, Irbesartan, Olmesartan, Telmisartan, and Valsartan, each ofwhich is also approved by the FDA for clinical use.

Accordingly, ARBs can be used treat hypertension; however, ARBs can beprescribed for other indications, as well. For example, Losartan canalso be used to reduce the risk of stroke in patients with hypertensionand left ventricular hypertrophy and/or to treat diabetic nephropathy inpatients with type 2 diabetes, an elevated serum creatinine andproteinuria, and a history of hypertension. Candesartan can be used inthe treatment of heart failure in adults with left ventricular systolicdysfunction to reduce the risk of death and/or hospitalizations due toheart failure. Irbesartan is indicated for the treatment of diabeticnephropathy in patients with type 2 diabetes, hypertension, and anelevated serum creatinine and proteinuria. Telmisartan can be used toreduce the risk myocardial infarction, stroke, or death fromcardiovascular causes in patients at high risk of developing majorcardiovascular events. Valsartan can be used for the treatment of heartfailure and used to reduce the risk of death in patients with leftventricular failure or left ventricular dysfunction following myocardialinfarction. The benefits of ARB treatment has also been tested for avariety of other diseases and disorders, including, e.g., congestiveheart failure, chronic heart failure, migraine, stroke, and renaldisease (Gales et al. (2010) Ann. Pharmacother. 44 (2): 360-6; Irie etal. (2012) Int J Cardiol. Published online Jul. 16, 2012; Kobori et al.(2012) Curr Pharm Des. Published online Nov. 21, 2012; Cancian et al.(2012) Eur J Gen Pract. Published on line Sep. 24, 2012). Each of theARBs described above is known by a number of trade names, the mostcommon of which are listed in Table 1 below.

TABLE 1 ARBs and Trade Names Drug Trade Names Azilsartan Edarbi ®Candesartan Blopress ®, Atacand ® Eprosartan Teveten ® IrbesartanAvapro ® Losartan Cozaar ®, Anzar ®, Arbloc ®, Angisartan ® Hartzar ®,Pharex ®, Neosartan ®, Hyoerthree ®, Getzar ®, Kenzar ®, Lozaris ®,Qxar ®, Normoten ®, Ecozar ®, Lifezar ® Olmesartan Olmezar ®, Olmetec ®Telmisartan Micardis ®, Pritor ®, Benicar ® Valsartan Diovan ®

In certain embodiments, methods described herein comprise administeringan ARB, and compositions and kits described herein comprise an ARB. Incertain embodiments, compositions and kits of the invention include anyARB, any combination of ARBs, or any prodrug, salt, or derivative of anyARB shown in Table 1.

Accordingly, in certain embodiments, the compositions of the inventioninclude Losartan and an antigen, vaccine, or anti-tumor preparation,where the Losartan in the composition is at a concentration sufficientto provide a dose of at least about 5 mg, at least about 10 mg, at leastabout 15 mg, at least about 20 mg, at least about 25 mg, at least about30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg,at least about 50 mg, at least about 60 mg, at least about 70 mg, atleast about 80 mg, of Losartan, including any range in between thesevalues, or less than about 50 mg, or less than about 25 mg of Losartan,including any range in between these values. In certain embodiments, theLosartan in the composition is at a concentration sufficient to providea dose of more than about 80 mg, more than about 100 mg, more than about125 mg, more than about 150 mg, more than about 175 mg, or more thanabout 200 mg or Losartan, including any range in between about 5 mg andabout 200 mg.

In certain embodiments, compositions of the invention can includeLosartan at a concentration sufficient to provide a dose of at leastabout 0.5 mg/kg, at least about 0.75 mg/kg, at least about 1.0 mg/kg, atleast about 1.25 mg·kg, at least about 1.5 mg/kg, at least about 1.75mg/kg, or at least about 2.0 mg/kg of Losartan, including any rangebetween about 0.5 mg/kg and about 1.75 mg/kg. In certain embodiments,compositions of the invention can include Losartan at a concentrationsufficient to provide a dose of more than about 1.75 mg/kg, at leastabout 2.0 mg/kg, at least about 5 mg/kg, at least about 7 mg/kg, atleast about 10 mg/kg, at least about 12 mg/kg, at least about 15 mg/kg,at least about 17 mg/kg, at least about 20 mg/kg, at least about 22mg/kg, at least about 25 mg/kg, at least about 27 mg/kg, or at leastabout 30 mg/kg or Losartan, including any range in between about 1.75mg/kg and about 30 mg/kg. In certain embodiments, the compositions ofthe invention can include Losartan at a concentration sufficient toprovide a dose of more than about 30 mg/kg, e.g., at least about 35mg/kg or at least about 40 mg/kg of Losartan, including any range inbetween about 30 mg/kg and about 40 mg/kg.

In certain embodiments, the invention provides compositions that caninclude Azilsartan and an antigen, vaccine, or anti-tumor preparation,where the Azilsartan in the composition is at a concentration sufficientto provide a dose of at least about 10 mg, at least about 20 mg, atleast about 40 mg, at least about 60 mg, at least about 70 mg, at leastabout 80 mg, of Losartan, including any range in between these values,or less than about 50 mg, or less than about 80 mg, including any rangebetween these values. In certain embodiments, the Azilsartan in thecomposition is at a concentration sufficient to provide a dose of morethan about 80 mg, more than about 100 mg, more than about 120 mg, morethan about 140 mg, or more than about 160 mg, including any range inbetween these values.

In certain embodiments, the invention also provides compositions thatcan include Candesartan and an antigen, vaccine, or anti-tumorpreparation, where the Candesartan in the composition is at aconcentration sufficient to provide a dose of at least about 0.5 mg, atleast about 1 mg, at least about 2 mg, at least about 3 mg, or less thanabout 4 mg, including any range between these values. In certainembodiments, the Candesartan in the composition is at a concentrationsufficient to provide a dose of more than about 32 mg, more than about40 mg, more than about 48 mg, more than about 56 mg, more than about 64mg or more than about 84 mg, including any range in between thesevalues. In certain embodiments, the Candesartan in the composition is ata concentration sufficient to provide a dose of less than about 32 mg,less than about 40 mg, less than about 48 mg, less than about 56 mg,less than about 64 mg or less than about 84 mg, including any range inbetween these values.

In certain embodiments, certain compositions of the invention caninclude Eprosartan and an antigen, vaccine, or anti-tumor preparation,where the Eprosartan in the composition is at a concentration sufficientto provide a dose of at least about 50 mg, at least about 100 mg, atleast about 200 mg, at least about 300 mg, at least about 400 mg, atleast about 500 mg, at least about 600 mg, at least about 700 mg, atleast about 800 mg, including any range between these values, or lessthan about 400 mg, less than about 300 mg, less than about 200 mg, orless than about 100 mg, including any range between these values. Incertain embodiments, the Eprosartan in the composition is at aconcentration sufficient to provide a dose of more than about 600 mg,more than about 750 mg, more than about 900 mg, more than about 1050 mg,or more than about 1200 mg, including any range in between these values.

In certain embodiments compositions of the invention can includeIrbesartan and an antigen, vaccine, or anti-tumor preparation, where theIrbesartan in the composition is at a concentration sufficient toprovide a dose of at least about 12.5 mg, at least about 25 mg, at leastabout 50 mg, at least about 75 mg, at least about 100 mg, at least about125 mg, at least about 150 mg, at least about 175 mg, at least about 200mg, at least about 250 mg, at least about 275 mg, or at least about 300mg, including any range between these values, or less than about 150 mg,less than about 100 mg, less than about 50 mg, including any rangebetween these values. In certain embodiments, the Irbesartan in thecomposition is at a concentration sufficient to provide a dose of morethan about 300 mg, more than about 375 mg, more than about 450 mg, morethan about 525 mg, or more than about 600 mg, including any range inbetween these values.

Alternatively, in other embodiments, compositions of the invention caninclude Olmesartan and an antigen, vaccine, or anti-tumor preparation,where the Olmesartan in the composition is at a concentration sufficientto provide a dose of at least about 5 mg, at least about 10 mg, at leastabout 15 mg, at least about 20 mg, at least about 25 mg, at least about30 mg, at least about 35 mg, at least about 40 mg, including any rangebetween these values, or less than about 20 mg, less than about 15 mg,less than about 10 mg including any range between these values. Incertain embodiments, the Olmesartan in the composition is at aconcentration sufficient to provide a dose of more than about 40 mg,more than about 50 mg, more than about 60 mg, more than about 70 mg, ormore than about 40 mg, including any range in between these values.

In certain embodiments, compositions of the invention can includeTelmisartan and an antigen, vaccine, or anti-tumor preparation, wherethe Telmisartan in the composition is at a concentration sufficient toprovide a dose of at least about 5 mg, at least about 10 mg, at leastabout 15 mg, at least about 20 mg, at least about 25 mg, at least about30 mg, at least about 35 mg, at least about 40 mg, including any rangebetween these values, or less than about 20 mg, less than about 15 mg,less than about 10 mg, including any range between these values. Incertain embodiments, the Telmisartan in the composition is at aconcentration sufficient to provide a dose of more than about 80 mg,more than about 100 mg, more than about 120 mg, more than about 140 mg,or more than about 160 mg, including any range in between these values.

In certain embodiments, compositions of the invention can includeValsartan and an antigen, vaccine, or anti-tumor preparation, where theValsartan in the composition is present at a concentration sufficient toprovide a dose of at least about 5 mg, at least about 10 mg, at leastabout 15 mg, at least about 20 mg, at least about 25 mg, at least about30 mg, at least about 35 mg, at least about 40 mg, including any rangebetween these values, or less than about 20 mg, less than about 15 mg,less than about 10 mg, including any range between these values. Incertain embodiments, the Valsartan in the composition is at aconcentration sufficient to provide a dose of more than about 320 mg,more than about 400 mg, more than about 480 mg, more than about 560 mg,or more than about 640 mg, including any range in between these values.

In some embodiments, the compositions comprise a mixture of 2 or moreARBs. In some aspects, the compositions may comprise about 2 to about 8,or about 2 to about 6, or about 2 to about 4, or 1, 2, 3, 4, 5, 6, 7, 8or more ARBs as described herein. In some embodiments, methods of thepresent invention comprise administering 2 or more ARBs. In some aspectsthe methods comprise administering about 2 to about 8, or about 2 toabout 6, or about 2 to about 4, or 1, 2, 3, 4, 5, 6, 7, 8 or more ARBsas described herein. Typically, ARBs are supplied in the form of tabletsfor oral administration. ARBs each exhibit different pharmacokineticproperties. For example, as shown below in Table 2, the biologicalhalf-lives and the bioavailability of ARBs vary widely, with Losartanhaving the lowest in vivo half-life.

TABLE 2 Comparison of ARB Pharmacokinetics Drug Biological Half-LifeBioavailability Azilsartan 11 hours  60% Candesartan 9 hours 15%Eprosartan 5 hours 13% Irbesartan 11-15 hours     70% Losartan 2 hours33% Olmesartan 14-16 hours     29% Telmisartan 24 hours  42-58%   Valsartan 6 hours 25%

Following administration (e.g., oral or otherwise), the presence and/orthe levels of an ARB (or of its metabolites) can be detected in anindividual's blood or urine using methods well known to those of skillin the art, including, for example, chromatographic and/or spectroscopictechniques. Details regarding such techniques are described in, e.g.,Nakashima et al. (1996) Blood Press. Suppl. 2: 62-66; Sica et al. (2005)Clin. Pharmacokinet. 44(8): 797-814; Lu et al. (2011) J. Pharm. Biomed.Anal. 54(1): 100-105; Yeung et al. (2000) Int. J. Pharmaceut. 204:17-22; Chando et al. (1998) Drug Metab. Dispos. 26(5): 408-417; McCarthyet al. (1998) J. Pharm Biomed. Anal. 17: 671-677; Ferreirós et al.(2007) Ther. Drug Monitoring 29(6): 824-834; Gonzalez et al. (2002) J.Chromatography A 949: 49-60; and others.

Compounds of Formula (I)-(V)

Certain embodiments of the invention relate to compounds of any one ofFormulae (I)-(V). In certain embodiments, methods described hereincomprise administration of compositions comprising a compound of any oneof Formulae (I)-(V), and compositions and kits described herein comprisea compound of any one of Formulae (I)-(V). Formulae (I)-(V) aredescribed below.

Formula (I)

The present disclosure provides a compound of Formula (I) andcompositions comprising a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen        or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆ alkyl;        or pharmaceutically acceptable salts thereof.

In some embodiments of Formula (I), R¹ is hydrogen. In some embodiments,R¹ is C₁₋₆ alkyl. In some embodiments, R¹ is methyl or ethyl. In someembodiments, R¹ is methyl.

In some embodiments of Formula (I),

is a single bond. In some embodiments,

is a double bond.

In some embodiments of Formula (I), Q¹ is N. In some embodiments, Q¹ isCH.

In some embodiments of Formula (I), R² is hydrogen. In some embodiments,R² is a C₁₋₆ alkylene, such as C₁ alkylene, C₂ alkylene, C₃ alkylene, C₄alkylene, C₅ alkylene, or C₆ alkylene. In some embodiments, R² is a C₁alkylene. In some embodiments, R² is C₁₋₆ alkylene, wherein one carbonunit of said alkylene is replaced with —O—, —S—, —SO—, —SO₂—, —NR^(a)—,or —CO—; wherein R^(a) is hydrogen or C₁₋₆ alkyl. In some embodiments,R² is C₁₋₆ alkylene, wherein one carbon unit of said alkylene isreplaced with —O—, —S—, —SO—, or —SO₂—. In some embodiments, R² is C₁₋₆alkylene, wherein one carbon unit of said alkylene is replaced with—NR^(a)—; wherein R^(a) is hydrogen or C₁₋₆ alkyl. In some embodiments,R² is C₁₋₆ alkylene, wherein one carbon unit of said alkylene isreplaced with —CO—.

In some embodiments of Formula (I), R³ is hydrogen. In some embodiments,R³ is an optionally substituted 5-membered heteroaryl ring; wherein theheteroaryl ring is optionally substituted with C₁₋₆ alkyl. In someembodiments, R³ is an optionally substituted 5-membered heteroaryl ringcontaining one, two, three, or four heteroatoms. In certain instances,the R³ heteroaryl ring contains one heteroatom. In certain instances,the R³ heteroaryl ring contains two heteroatoms. In certain instances,the R³ heteroaryl ring contains three heteroatoms. In certain instances,the R³ heteroaryl ring contains four heteroatoms. In certain instances,the R³ heteroaryl ring contains at least one heteroatom selected fromnitrogen, sulfur, and oxygen. In certain instances, the R³ heteroarylring contains two heteroatoms selected from nitrogen, sulfur, andoxygen. In certain instances, the R³ heteroaryl ring contains twonitrogen heteroatoms. In certain instances, the R³ heteroaryl ringcontains carbon, nitrogen, and sulfur ring members. In certaininstances, the R³ heteroaryl ring contains carbon and nitrogen ringmembers. In certain instances, the R³ heteroaryl ring contains carbon,nitrogen, and oxygen ring members.

In certain instances, the R³ heteroaryl ring is furanyl, thiophenyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, thiadiazolyl, oxadiazolyl,triazolyl, or tetrazolyl, each optionally substituted with C₁₋₆alkyl, asdescribed for the R³ heteroaryl ring. In certain instances, the R³heteroaryl ring is selected from the following:

each optionally substituted with C₁₋₆ alkyl, as described for the R³heteroaryl ring. In certain instances, the R³ heteroaryl ring is

Formula (II)

The present disclosure provides a compound of Formula (II) andcompositions comprising a compound of Formula (II):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof

In some embodiments of Formula (II), R¹ is hydrogen. In someembodiments, R¹ is C₁₋₆ alkyl. In some embodiments, R¹ is methyl orethyl. In some embodiments, R¹ is methyl.

In some embodiments of Formula (II),

is a single bond. In some embodiments,

is a double bond.

In some embodiments of Formula (II), Q¹ is N. In some embodiments, Q¹ isCH.

In some embodiments of Formula (II), R⁴ is an optionally substituted5-membered heteroaryl ring; wherein the heteroaryl ring is optionallysubstituted with C₁₋₆alkyl. In some embodiments, R⁴ is an optionallysubstituted 5-membered heteroaryl ring containing one, two, three, orfour heteroatoms. In certain instances, the R⁴ heteroaryl ring containsone heteroatom. In certain instances, the R⁴ heteroaryl ring containstwo heteroatoms. In certain instances, the R⁴ heteroaryl ring containsthree heteroatoms. In certain instances, the R⁴ heteroaryl ring containsfour heteroatoms. In certain instances, the R⁴ heteroaryl ring containsat least one heteroatom selected from nitrogen, sulfur, and oxygen. Incertain instances, the R⁴ heteroaryl ring contains two heteroatomsselected from nitrogen, sulfur, and oxygen. In certain instances, the R⁴heteroaryl ring contains two nitrogen heteroatoms. In certain instances,the R⁴ heteroaryl ring contains carbon, nitrogen, and sulfur ringmembers. In certain instances, the R⁴ heteroaryl ring contains carbonand nitrogen ring members. In certain instances, the R⁴ heteroaryl ringcontains carbon, nitrogen, and oxygen ring members.

In certain instances, the R⁴ heteroaryl ring is furanyl, thiophenyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, thiadiazolyl, oxadiazolyl,triazolyl, or tetrazolyl, each optionally substituted with C₁₋₆ alkyl,as described for the R⁴ heteroaryl ring. In certain instances, the R⁴heteroaryl ring is selected from the following:

each optionally substituted with C₁₋₆alkyl, as described for the R³heteroaryl ring. In certain instances, the R⁴ heteroaryl ring is

Formula (III)

The present disclosure provides a compound of Formula (III) andcompositions comprising a compound of Formula (III):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is an optionally substituted 5-membered heteroaryl ring;        wherein the heteroaryl ring is optionally substituted with C₁₋₆        alkyl;        or pharmaceutically acceptable salts thereof

In some embodiments of Formula (III),

is a single bond. In some embodiments,

is a double bond.

In some embodiments of Formula (III), Q¹ is N. In some embodiments, Q¹is CH.

In some embodiments of Formula (III), R⁴ is an optionally substituted5-membered heteroaryl ring; wherein the heteroaryl ring is optionallysubstituted with C₁₋₆alkyl. In some embodiments, R⁴ is an optionallysubstituted 5-membered heteroaryl ring containing one, two, three, orfour heteroatoms. In certain instances, the R⁴ heteroaryl ring containsone heteroatom. In certain instances, the R⁴ heteroaryl ring containstwo heteroatoms. In certain instances, the R⁴ heteroaryl ring containsthree heteroatoms. In certain instances, the R⁴ heteroaryl ring containsfour heteroatoms. In certain instances, the R⁴ heteroaryl ring containsat least one heteroatom selected from nitrogen, sulfur, and oxygen. Incertain instances, the R⁴ heteroaryl ring contains two heteroatomsselected from nitrogen, sulfur, and oxygen. In certain instances, the R⁴heteroaryl ring contains two nitrogen heteroatoms. In certain instances,the R⁴ heteroaryl ring contains carbon, nitrogen, and sulfur ringmembers. In certain instances, the R⁴ heteroaryl ring contains carbonand nitrogen ring members. In certain instances, the R⁴ heteroaryl ringcontains carbon, nitrogen, and oxygen ring members.

In certain instances, the R⁴ heteroaryl ring is furanyl, thiophenyl,pyrrolyl, oxazolyl, thiazolyl, imidazolyl, thiadiazolyl, oxadiazolyl,triazolyl, or tetrazolyl, each optionally substituted with C₁₋₆alkyl, asdescribed for the R⁴ heteroaryl ring. In certain instances, the R⁴heteroaryl ring is selected from the following:

each optionally substituted with C₁₋₆alkyl, as described for the R³heteroaryl ring. In certain instances, the R⁴ heteroaryl ring is

Formula (IV)

The present disclosure provides a compound of Formula (IV) andcompositions comprising a compound of Formula (IV):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆ alkyl;    -   is a single bond or double bond;    -   Q is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments of Formula (IV), R¹ is hydrogen. In someembodiments, R¹ is C₁₋₆ alkyl. In some embodiments, R¹ is methyl orethyl. In some embodiments, R¹ is methyl.

In some embodiments of Formula (IV),

is a single bond. In some embodiments,

is a double bond.

In some embodiments of Formula (IV), Q¹ is N. In some embodiments, Q¹ isCH.

In some embodiments of Formula (IV), R⁴ is

In some embodiments, R⁴ is

Formula (V)

The present disclosure provides a compound of Formula (V) andcompositions comprising a compound of Formula (V):

-   -   wherein    -   is a single bond or double bond;    -   Q¹ is N or CH; and    -   R⁴ is selected from

or pharmaceutically acceptable salts thereof.

In some embodiments of Formula (V),

is a single bond. In some embodiments,

is a double bond.

In some embodiments of Formula (V), Q¹ is N. In some embodiments, Q¹ isCH.

In some embodiments of Formula (V), R⁴ is

In some embodiments, R⁴ is

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination. All combinations of the embodimentspertaining to the chemical groups represented by the variables arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed, to the extent that such combinations embrace compounds thatare stable compounds (i.e., compounds that can be isolated,characterized, and tested for biological activity). In addition, allsubcombinations of the chemical groups listed in the embodimentsdescribing such variables are also specifically embraced by the presentinvention and are disclosed herein just as if each and every suchsub-combination of chemical groups was individually and explicitlydisclosed herein.

Particular compounds of interest are shown below:

Ondansetron;

-   9-methyl-3-((2-methyl-1H-imidazol-1-yl)methyl)-2,3-dihydro-1H-carbazol-4(9H)-one    Alosetron;-   5-methyl-2-(4-methyl-1H-imidazol-5-yl)methyl)-2,3,4,4a,5,9b-hexahydro-1H-pyrido[4,3-1]indol-1-one

Alosetron can be used in the management of severe diarrhea-predominantirritable bowel syndrome (IBS). Ondansetron can be prescribed to treatand/or prevent chemotherapy-induced nausea and vomiting (CINV).Ondansetron has been indicated in the prevention and treatment ofradiation-induced nausea and vomiting (RINV), and post-operative nauseaand vomiting (PONV). The benefits of Ondansetron treatment have alsobeen tested for a variety of other diseases and disorders, including,e.g., motion sickness (Levine et al. (2000) Aviat Space Environ Med. 71:1111-1114; Muth et al. (2007) Aviat Space Environ Med. 78: 686-92);lesional vestibular disorder (European Patent Application No. 2432467 A1and US Patent Application Publication 2012/0064094); anti-psychoticinduced tardive dyskinesia in people with schizophrenia (Zullino et al.(2001) Am. J. Psychiatry 158: 657-8 and Sirota et al. (2000) Am. J.Psychiatry 157: 287-289); and schizophrenia (Zhang et al. (2006) SchizRes 88: 102-110). Other medical conditions that may be treated usingondansetron include, e.g., gastroenteritis, pediatric gastroenteritis,opioid-induced nausea, nausea and vomiting of pregnancy, andobsessive-compulsive disorder (Broocks et al. (1998) Psychiatry Res. 79:11-20).

Ondansetron and Alosetron are each known by a number of trade names, themost common of which are listed in Table 3 below.

TABLE 3 Trade Names Trade Names Alosetron Lotronex ® OndansetronDoran ®, Lupisetron ®, Mylan⁻Ondansetron, Myset ®, Ranidom ®,Vomicare ®, Vomiven ®, Zofran ®, Zofran ODT ®, Zuplenz ®

Accordingly, in certain embodiments, the compositions of the inventioninclude Ondansetron and an antigen, vaccine, or anti-tumor preparation.In certain embodiments, the composition is administered orally. Incertain embodiments, Ondansetron in the orally administered compositionis at a concentration sufficient to provide a dose of at least about 0.5mg, at least about 1 mg, at least about 2 mg, at least about 3 mg, atleast about 4 mg, at least about 5 mg, at least about 6 mg, at leastabout 7 mg, at least about 8 mg, at least about 10 mg, or less thanabout 12 mg, or less than about 18 mg, or less than about 24 mg ofOndansetron, including any range in between these values. In certainembodiments, the Ondansetron in the orally administered composition isat a concentration sufficient to provide a dose of more than about 24mg, more than about 26 mg, more than about 28 mg, more than about 30 mg,more than about 32 mg, or more than about 34 mg Ondansetron, includingany range in between these values.

In certain embodiments, compositions of the invention are administeredintravenously. In certain embodiments, compositions for intravenousadministration can include Ondansetron at a concentration sufficient toprovide a dose of at least about 0.5 mg/kg/day, at least about 0.75mg/kg/day, at least about 1.0 mg/kg/day, at least about 1.5 mg/kg/day,at least about 2.0 mg/kg/day, at least about 2.5 mg/kg/day, at leastabout 3.0 mg/kg/day, at least about 3.5 mg/kg/day, at least about 4.0mg/kg/day, at least about 4.5 mg/kg/day, at least about 5.0 mg/kg/day,at least about 5.5 mg/kg/day, at least about 6.0 mg/kg/day, at leastabout 6.5 mg/kg/day, at least about 7.0 mg/kg/day, at least about 7.5mg/kg/day, at least about 8.0 mg/kg/day, or less than about 8.5mg/kg/day of Ondansetron, including any range between these values.

In certain embodiments, the invention provides compositions that caninclude Alosetron and an antigen, vaccine, or anti-tumor preparation,where the Alosetron in the composition is at a concentration sufficientto provide a dose of at least about 0.1 mg, at least about 0.2 mg, atleast about 0.3 mg, at least about 0.4 mg, at least about 0.6 mg, atleast about 0.8 mg, at least about 0.9 mg, at least about 1 mg,including any range between these values, or less than about 0.5 mg orless than about 0.7 mg or less than about 0.9 mg, or less than about 1mg of Alosetron, including any range between these values. In certainembodiments, the Alosetron in the composition is at a concentrationsufficient to provide a dose of more than about 2 mg, more than about 3mg, more than about 4 mg, more than about 5 mg, or more than about 6 mgAlosetron, including any range in between these values.

Ondansetron and Alosetron are supplied in the form of tablets orsolutions for oral administration. In one embodiment, Ondansetron isalso supplied in solution form for parenteral administration.Ondansetron and Alosetron each exhibit different pharmacokineticproperties. For example, the biological half-life of Alosetron is about1.5 to about 1.7 hours, and the biological half-life of Ondansetron isabout 3.9 hours.

Following administration (e.g., oral, intramuscular, subcutaneous, orotherwise), the presence and/or the levels of a compound of Formula (I)(or of its metabolites) can be detected in an individual's blood orurine using methods well known to those of skill in the art, including,for example, chromatographic and/or spectroscopic techniques. Detailsregarding such techniques are described in, e.g., Somers et al. (2007)Xenobiotica 37: 855-869; Koch et al. (2002) Br J Clin Pharmacol. 53:238-242; Xu et al. (2000) J Mass Spectrom 35: 1329-1334; Roila et al.(1995) Clin Pharmacokinet 29: 95-103; and others.

Antigens

In certain embodiments, compositions and kits of the invention includean antigen, and certain methods of the invention comprise administeringan antigen. In certain embodiments, the antigen present in thecompositions provided by the invention can be any material or substancethat can induce an immune response (i.e., cellular and/or humoral immuneresponse) by the immune system of a human or animal. For example, theantigen can be a polypeptide of interest derived from an infectiousagent, e.g., a bacterium, a virus, a fungus, a protozoan, a parasite, ora prion. In particular embodiments, the antigen can be a whole microbeor a mixture thereof, and the compositions can include a live wholeinfectious agent. In certain embodiments, the compositions can include akilled or inactivated (attenuated) infectious agent.

In certain embodiments, the antigen comprises, e.g., a polypeptide,nucleic acid, polysaccharide, a fatty acid or the like, derived from aninfectious agent. In certain embodiments, the antigen can be a subunitor fragment of a polypeptide, or a fragment of a nucleic acid orpolysaccharide derived from an infectious agent. In certain embodiments,the antigen is a recombinant polypeptide produced in a heterologousexpression system, e.g., a recombinant protein derived from aninfectious agent that was expressed in and purified from cells ofanother organism. However, in particular embodiments, an antigen canalso be a recombinant nucleic acid construct which encodes a polypeptideantigen of interest (e.g., an expression construct). In certainembodiments, the antigen can comprise a viral subunit, a virus-likeparticle, a capsular (poly) saccharide; a bacterial outer membrane blebformation containing one or more of bacterial outer membrane proteins, aphospholipid, a lipopolysaccharide, or a polysaccharide.

In certain embodiments, the antigen can be a naturally occurringsubstance. In certain embodiments, the antigen comprises or is derivedfrom an allergen, e.g., pollen. In certain embodiments, the antigencomprises or is derived from a toxin. In certain embodiments, theantigen comprises or is derived from an addictive substance, including,without limitation, nicotine, caffeine, alcohol, and the like. Incertain embodiments, the antigen can be a non-naturally occurring (i.e.,synthetic) substance, e.g., a synthetic peptide, a syntheticpolysaccharide, or a synthetic polymer.

In certain embodiments, the antigen can be provided in a vaccine, e.g.,any vaccine known in the art. For example, the vaccine can be a nucleicacid construct (e.g., a DNA vaccine) or the vaccine can be a viralvector vaccine, which uses live viruses to carry DNA into anindividual's cells. The DNA contained in the viral vaccine encodesantigen(s) that, once expressed in the infected cells, elicit an immuneresponse. Alternatively, in certain embodiments, the vaccine can be asubunit vaccine, e.g., a specific protein from a virus. In certainembodiments, the vaccine can be a dendritic cell vaccine, in which anindividual's dendritic cells are cultured with an antigen and thenre-injected into the individual to stimulate an immune response. Incertain embodiments, the vaccine can be a monovalent vaccine, i.e.,containing a single antigen. In certain embodiments, the vaccinecontaining the antigen is a polyvalent or multivalent vaccine, i.e.,containing more than one antigen.

Pharmaceutically Acceptable Compositions and Formulations

The compositions or formulations of the present invention include anycomposition or formulation described herein. The various compounds(e.g., the ARBs, the compounds of Formulae (I)-(V), the antigens, thevaccines, and the anti-tumor compositions) described herein may bepresent in various compositions or formulations, including thosesuitable for administration to an individual (e.g., pharmaceuticalcompositions). In some embodiments, a composition of the presentinvention includes any one of the combinations of compounds listed inTable 4, herein. The compositions of the invention can be incorporatedinto a variety of formulations for therapeutic administration bycombination with appropriate pharmaceutically acceptable carriers ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Pharmaceutical compositions can include,depending on the formulation desired, pharmaceutically-acceptable,non-toxic carriers or diluents, which are defined as vehicles commonlyused to formulate pharmaceutical compositions for animal or humanadministration. The carrier or diluent is selected so as not to affectthe biological activity of the combination. Examples of such carriers ordiluents are distilled water, buffered water, physiological saline, PBS,Ringer's solution, dextrose solution, and Hank's solution. In addition,the pharmaceutical composition or formulation can include othercarriers, adjuvants, or non-toxic, nontherapeutic, non-immunogenicstabilizers, excipients and the like. The compositions can also includeadditional substances to approximate physiological conditions, such aspH adjusting and buffering agents, toxicity adjusting agents, wettingagents and detergents.

The composition can also include any of a variety of stabilizing agents,such as an antioxidant for example. When the pharmaceutical compositionincludes a polypeptide, the polypeptide can be complexed with variouswell-known compounds that enhance the in vivo stability of thepolypeptide, or otherwise enhance its pharmacological properties (e.g.,increase the half-life of the polypeptide, reduce its toxicity, andenhance solubility or uptake). Examples of such modifications orcomplexing agents include sulfate, gluconate, citrate and phosphate. Thepolypeptides of a composition can also be complexed with molecules thatenhance their in vivo attributes. Such molecules include, for example,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

Further guidance regarding formulations that are suitable for varioustypes of administration can be found in Remington's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed. (1985).For a brief review of methods for drug delivery, see, Langer, Science249:1527-1533 (1990).

For oral administration, the compositions of the invention can beadministered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. The active component(s) can be encapsulated in gelatincapsules together with inactive ingredients and powdered carriers, suchas glucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate. Examples of additional inactive ingredients thatmay be added to provide desirable color, taste, stability, bufferingcapacity, dispersion or other known desirable features are red ironoxide, silica gel, sodium lauryl sulfate, titanium dioxide, and ediblewhite ink. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

Formulations suitable for parenteral administration include, e.g.,aqueous and non-aqueous, isotonic sterile injection solutions, which cancontain antioxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

The components used to formulate the pharmaceutical compositions arepreferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Kits

The invention also provides kits and articles of manufacture comprisingcompositions described herein for use in the methods of enhancing animmune response, decreasing recruitment of monocytes to a lymph node,amplifying vaccine immunity, and inhibiting tumor growth and metastasis.In certain embodiments, kits of the invention include a container orcompartment comprising a compound of Formula (I):

-   -   wherein    -   R¹ is hydrogen or C₁₋₆alkyl;    -   is a single bond or double bond;    -   Q¹ is N or CH;    -   R² is selected from hydrogen and C₁₋₆ alkylene, wherein one        carbon unit of said alkylene is optionally replaced with —O—,        —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—; wherein R^(a) is hydrogen    -   or C₁₋₆ alkyl; and    -   R³ is hydrogen or an optionally substituted 5-membered        heteroaryl ring; wherein the heteroaryl ring is optionally        substituted with C₁₋₆alkyl;        or pharmaceutically acceptable salts thereof, and an antigen, a        vaccine, or anti-tumor preparation. In certain embodiments, the        kit includes Ondansetron. In certain embodiments, the kit        includes Alosetron. In certain embodiments, kits of the        invention provide the compound of Formula (I) (e.g., Ondansetron        and/or Alosetron) in a first container or compartment and the        antigen, vaccine, or anti-tumor preparation in a second        container or compartment.

In certain embodiments, kits of the invention include a container orcompartment comprising an ARB and an antigen, a vaccine, or anti-tumorpreparation. In certain embodiments, kits of the invention provide theARB in a first container or compartment and the antigen, vaccine, oranti-tumor preparation in a second container or compartment.

In certain embodiments, the kits of the invention comprise a singlecontainer comprising both a compound of Formula (I) (e.g., Ondansetronand/or Alosetron) and an antigen, vaccine, or anti-tumor preparation. Incertain embodiments, the kits of the invention comprise a singlecontainer comprising both an ARB and an antigen, vaccine, or anti-tumorpreparation. In one embodiment, the kit of the invention comprisesLosartan and a tyrosine kinase inhibitor.

In one certain embodiment, a kit of the invention comprises a containercomprising a composition (e.g., a pharmaceutically acceptablecomposition or formulation) comprising both a Compound of Formula (I)and an antigen, vaccine, or anti-tumor preparation.

In one certain embodiment, a kit of the invention comprises a containercomprising a composition (e.g., a pharmaceutically acceptablecomposition or formulation) comprising both an ARB and an antigen,vaccine, or anti-tumor preparation.

In particular embodiments, the combination of (i) ARB or Compound ofFormula (I) and (ii) antigen, vaccine or anti-tumor preparation, presentin a kit or composition of the present invention (including compositionspresent within a kit of the present invention) is selected from any ofthe combinations listed in Table 4 below. In one certain embodiment, thekit of the invention comprises at least two separate containers, witheach separate container comprising at least one of two separatecompositions (e.g., pharmaceutically acceptable compositions orformulations), wherein a first of the two compositions comprises an ARBor a Compound of Formula (I) and the second of the two separatecompositions comprises an antigen, vaccine, or anti-tumor preparation.In particular embodiments, the combination of the first and the secondcompositions is selected from any one of the combinations listed inTable 4.

TABLE 4 Combinations of monocyte migration inhibitors with antigens,vaccines, or anti-tumor preps Column A: Column B: Column C: CombinationARB or Compound Antigen, Vaccine, or Number of Formula (I) Anti-tumorpreparation 1 Ondansetron Antigen 2 Ondansetron Vaccine 3 OndansetronAnti-tumor preparation 4 Ondansetron Sunitinib 5 Alosetron Antigen 6Alosetron Vaccine 7 Alosetron Anti-tumor preparation 8 Alosetron Antigen9 Azilsartan Antigen 10 Azilsartan Vaccine 11 Azilsartan Anti-tumorpreparation 12 Azilsartan Sunitinib 13 Candesartan Antigen 14Candesartan Vaccine 15 Candesartan Anti-tumor preparation 16 CandesartanSunitinib 17 Eprosartan Antigen 18 Eprosartan Vaccine 19 EprosartanAnti-tumor preparation 20 Eprosartan Sunitinib 21 Irbesartan Antigen 22Irbesartan Vaccine 23 Irbesartan Anti-tumor preparation 24 IrbesartanSunitinib 25 Losartan Antigen 26 Losartan Vaccine 27 Losartan Anti-tumorpreparation 28 Losartan Sunitinib 29 Olmesartan Antigen 30 OlmesartanVaccine 31 Olmesartan Anti-tumor preparation 32 Olmesartan Sunitinib 33Telmisartan Antigen 34 Telmisartan Vaccine 35 Telmisartan Anti-tumorpreparation 36 Telmisartan Sunitinib 37 Valsartan Antigen 38 ValsartanVaccine 39 Valsartan Anti-tumor preparation 40 Valsartan Sunitinib

In certain embodiments, the kits of the invention further compriseinstructions for use in accordance with any of the methods describedherein.

Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable. Theinstructions relating to the use of the compositions described hereingenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. In some embodiments, theinstructions comprise instructions for administering an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) inconjunction with an antigen, a vaccine, or an anti-tumor preparation.Kits of the invention may further comprise a description of selecting anindividual suitable or treatment.

The present invention also provides kits comprising an ARB, a compoundof Formula (I), or a compositions described herein and furthercomprising instruction(s) on methods of using the composition or kit,such as uses further described herein. The kit may comprise one or moreunit dosages of an ARB, a compound of Formula (I), or a compositiondescribed herein. The kit may contain instructions for administering theARB or the compound of Formula (I) (e.g., Ondansetron and/or Alosetron)and the antigen, vaccine, or anti-tumor preparation simultaneously orsequentially, as described elsewhere herein. In some embodiments, thekit of the invention comprises the packaging described herein. In otherembodiments, the kit of the invention comprises the packaging describedherein and a second packaging comprising a buffer. The kit may furtherinclude other materials desirable from a commercial and user standpoint,including other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for performing any methods describedherein.

In certain embodiments, kits may also include multiple unit doses of anARB or a compound of Formula (I) (e.g., Ondansetron and/or Alosetron)and an antigen, a vaccine, or an anti-tumor preparation and instructionsfor use and packaged in quantities sufficient for storage and use inpharmacies, for example, hospital pharmacies and compounding pharmacies.In certain embodiments, the kits of the invention are in suitablepackaging. Suitable packaging includes, but is not limited to, vials,bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags),and the like. In certain embodiments, a container or compartment isvials, bottles, jars, flexible packaging (e.g., sealed mylar or plasticbags), and the like. Kits may optionally provide additional componentssuch as buffers and interpretative information.

Methods of Enhancing an Immune Response Against an Antigen in anIndividual

A vaccine can be administered to an individual to elicit an immuneresponse that can lessen the severity and/or duration of a disease orinfection. Vaccines can include antigens. However, not all antigens arecapable of stimulating a sufficiently robust B cell and/or T cellresponse to produce protective immunity. Certain individuals, forexample, children, the elderly, or the immunocompromised, may not becapable of mounting a robust immune response. The methods andcompositions described herein can be used to enhance an immune responsein an individual to whom an antigen has been administered. Suchmethods/uses include administering a composition containing the antigento the individual in conjunction with an ARB or a compound of Formula(I) (e.g., Ondansetron and/or Alosetron).

Accordingly, in one embodiment, the present invention includes a methodof enhancing an immune response to an antigen in an individual,comprising administering to the individual an antigen and an ARB. Inanother embodiment, the present invention includes a method of enhancingan immune response to an antigen in an individual, comprisingadministering to the individual an antigen and a compound of Formula(I). In certain embodiments, the ARB or compound of formula (I) areadministered in an effective amount, e.g., an amount sufficient toenhance the immune response to the antigen. In particular embodiments,the antigen, the ARB and/or the compound of Formula (I) are present inone or more composition, e.g., one or more pharmaceutical compositions.In certain embodiments, the methods include administering a compositioncontaining the antigen to the individual with Ondansetron. In certainembodiments, the methods include administering a composition containingthe antigen to the individual with Alosetron. In certain embodiments,the methods include administering a composition containing the antigento the individual in conjunction with an ARB (e.g., Losartan,Candesartan, Eprosartan, Irbesartan, Olmesartan, Telmimsartan,Azilsaran, and/or Valsartan).

The compound of Formula (I) administered in conjunction with the antigencan be any compound, combination of compounds, or any prodrug, salt, orderivative of a compound described herein. The ARB administered inconjunction with the antigen can be any ARB, combination of ARBs, or anyprodrug, salt, or derivative of an ARB described herein. The antigenadministered in conjunction with the ARB or the compound of Formula (I)(e.g., Ondansetron or Alosetron) can be any antigen described herein.

In certain embodiments, the combination of antigen and ARB or compoundof Formula (I) is any described herein (e.g., any ARB described hereincombined with any antigen described herein; or e.g., any compound ofFormula (I) described herein combined with any antigen describedherein). The ARB or the compound of Formula (I) (e.g., Ondansetron orAlosetron) and the antigen can be present in a single pharmaceuticalcomposition, or they can be provided in separate compositions that canbe administered in any order relative to one another or administeredsimultaneously, as described herein. Compositions containing the ARB orthe compound of Formula (I) (e.g., Ondansetron or Alosetron) and/or theantigen can be administered according to any method known in the art atdosages described elsewhere herein.

In certain embodiments, the individuals to whom a compound of Formula(I) can be administered in conjunction with the antigen can be those whoare otherwise not receiving the Ondansetron or Alosetron for thetreatment of, e.g., irritable bowel syndrome (IBS), post-operativenausea and vomiting (PONV), chemotherapy-induced nausea and vomiting(CINV), radiation-induced nausea and vomiting (RINV), and/or otherconditions, as described herein. In some embodiments, an individual whohas a condition or disease described herein, or is otherwise in need oftreatment, can receive Ondansetron and/or Alosetron in conjunction withan antigen for the purpose of enhancing an immune response if theindividual is not being treated with Ondansetron and/or Alosetron.Alternatively, in other embodiments, the individuals to whom a compoundof Formula (I) is administered in conjunction with the antigen can bethose who have temporarily suspended Ondansetron and/or Alosetrontreatment and have been shown, using methods well known in the art, tonot have detectable levels of Ondansetron and/or Alosetron in theirblood and/or urine prior to the administration of the Ondansetron and/orAlosetron in conjunction with the antigen. Details regarding suchtechniques are described in, e.g., Somers et al. (2007) Xenobiotica 37:855-869; Koch et al. (2002) Br J Clin Pharmacol. 53: 238-242; Xu et al.(2000) J Mass Spectrom 35: 1329-1334; Roila et al. (1995) ClinPharmacokinet 29: 95-103; and others.

In certain embodiments, the individuals to whom an ARB is administeredin conjunction with an antigen can be those who are otherwise notreceiving an ARB for treatment of a pre-existing condition, e.g.,hypertension, diabetic nephropathy, heart failure, and/or otherconditions, as described herein. In some embodiments, an individual whohas a condition or disease described herein, or who is otherwise in needof treatment, can receive an ARB in conjunction with an antigen for thepurpose of enhancing an immune response if the individual is not beingtreated with an ARB. Alternatively, in certain embodiments, theindividuals to whom an ARB is administered in conjunction with anantigen can be those who have temporarily suspended ARB treatment andhave been shown, using methods well known in the art, to not havedetectable levels of an ARB in their blood and/or urine prior to theadministration of the ARB in conjunction with the antigen. Detailsregarding such techniques are described in, e.g., Nakashima et al.(1996) Blood Press. Suppl. 2: 62-66; Sica et al. (2005) Clin.Pharmacokinet 44(8): 797-814; Lu et al. (2011) J. Pharm. Biomed. Anal.54(1): 100-105; Yeung et al. (2000) Int. J. Pharmaceut. 204: 17-22;Chando et al. (1998) Drug Metab. Dispos. 26(5): 408-417; McCarthy et al.(1998) J. Pharm Biomed. Anal. 17: 671-677; Ferreirós et al. (2007) Ther.Drug Monitoring 29(6): 824-834; González et al. (2002) J. ChromatographyA 949: 49-60; and others.

Detecting Enhanced Immune Responses

In certain embodiments, the methods of the invention can be used toenhance a humoral immune response (i.e., B cell response) and/or acellular response (i.e., T cell response). An enhanced humoral immuneresponse can be demonstrated by showing that the antibody titers againsta specific antigen from, e.g., an individual who received an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) inconjunction with the antigen, are higher than antibody titers againstthe antigen from, e.g., an individual who received only the antigen.Antibody levels against a specific antigen can be determined via solidphase radioimmunoassay (RIA), in which serially diluted blood serum isincubated in microtiter wells previously coated with the antigenadministered to the individual in conjunction with an ARB or a compoundof Formula (I) (e.g., Ondansetron and/or Alosetron). Bound antibody isdetected by employing 125I-labeled anti-immunoglobulin antibodies. Theamount of specific antibody in the antiserum is then determined from astandard curve generated with a specific antibody of knownconcentration. Another method by which antibody titers can be determinedis ELISA, in which an enzyme-conjugated secondary antibody, rather thanradiolabeled secondary antibody, is used to detect the binding ofprimary antibodies to the antigen. Such methods are well known to thoseof skill in the art and are described in further detail in, e.g.,Essentials of Immunology & Serology (Stanley, 2002); Clinical Immunology& Serology: A Laboratory Perspective (Stevens, 2009); ContemporaryClinical Immunology and Serology (Rittenhouse-Olson and DeNardin, 2012);Cooper et al. (2001) Curr Protoc Mol Biol. Chapter 11: Unit 11.17;Player et al. (1993) J Virol. Methods 45(1): 67-72; Gheesling et al.(1994) J Clin. Microbiol. 32(6): 1475-82; and others. Additionally, manycommercial kits and automated systems are currently available, e.g.,from Abcam, Imgenex, Cygnus, and others, to detect antigen-specificantibodies following vaccination.

An enhanced cellular immune response can be demonstrated by showing thatT cells from, e.g., an individual who received an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) in conjunction with aspecific antigen, are more highly activated by the antigen than T cellsfrom, e.g., an individual who received only the antigen. One of the mostcommon ways to assess T cell activation is to measure T-cellproliferation or T-cell cytokine elaboration upon in vitro stimulationof T-cells by the antigen administered in the methods (e.g., wholeantigen or fragments thereof). This can be assessed via, e.g., ELISpot,a sensitive colorimetric assay based on ELISA that can detect secretedcytokine at the single cell level, rather than antigen-bound antibody.Details regarding ELISpot and related techniques (such as FLUROspot) aredescribed in, e.g., Tobery et al. (2001) J. Immunol. Meth. 254: 59-66;Braun et al. (2006) Virology J. 3:53-68; Davis, et al. (2004) PNAS101(29) 10697-10702; Posavad, et al. (2011) Vaccine 29(40): 7058-7066;Hutchings et al. (1989) J Immunol Methods. 120: 1-8; and others. ELISpotkits are also commercially available from, e.g., MABTECH AB, R&DSystems, BD Biosciences, ABCam, and other manufacturers.

T cell activation can also be assayed via flow cytometry. For example,T-cells can be stimulated with, e.g., a protein, peptide, or group ofpeptides derived from the antigen administered during the methods, andcultured for a period of time. During that time, an inhibitor is addedwhich blocks the release of cytokines from the T cells. The T cells arethen fixed and permeabilized to allow anti-cytokine-specific antibodiesto stain the intracellular cytokines, allowing them to be visualizedduring FACS analysis in relatively high quantity. Further detailsregarding this technique are described in, e.g., Caruso et al. (1997)Cytometry 27(1): 71-76; Nomura et al. (2000) Cytometry. 40: 60-68; Suniet al. (1998) J Immunol Methods. 212: 89-98; and others.

In particular embodiments, an individual to whom an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) is administered inconjunction with an antigen can exhibit a humoral immune response thatis at least about 2-fold, at least about 5-fold, at least about 10-fold,at least about 20-fold, at least about 50-fold, at least about 60-fold,at least about 70-fold, at least about 80-fold, at least about 90 fold,at least about 100-fold, at least about 110-fold, at least about125-fold, at least about 150-fold, at least about 175-fold, at leastabout 200-fold, or more than 200-fold (e.g., about 250-fold, about300-fold, or about 350-fold) higher than the humoral immune responseexhibited by an individual to whom vaccine alone is administered,including any range in between about 2-fold and about 300-fold. Anindividual to whom an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) is administered in conjunction with anantigen can exhibit a cellular immune response that is at least about1.2-fold, at least about 1.5-fold, at least about 2-fold, at least about2.5-fold, at least about 3-fold, at least about 3.5 fold, at least about4-fold, at least about 4.5-fold, at least about 5-fold, or more thanabout 5-fold (e.g., about 5.5-fold, about 6-fold, or about 6.5-fold)higher than the cellular immune response exhibited by an individual towhom vaccine alone is administered, including any range between about1.2-fold and about 6.5 fold.

Antigens

In particular embodiments, the antigen is administered in conjunctionwith an ARB or a compound of Formula (I) (e.g., Ondansetron and/orAlosetron) in any suitable amount that is sufficient to generate anenhanced immune response. In certain embodiments, the antigenadministered in conjunction with the ARB or a compound of Formula (I)(e.g., Ondansetron and/or Alosetron) can be any antigen or combinationof antigens described herein. The amount of antigen to be included inthe compositions and used in the methods of the present invention (i.e.,any of the methods described herein) will depend on the immunogenicityof the antigen itself and the efficacy of any adjuvants co-administeredtherewith. In general, an immunologically effective dose comprisesbetween about 1 μg to about 1000 μg of the antigen, preferably betweenabout 5 μg to about 500 μg, more preferably between about 10 μg to about200 μg. In some embodiments, an immunologically effective dose can be atleast about 1 μg, at least about 5 μg, at least about 10 μg, at leastabout 25 μg, at least about 50 μg, at least about 100 μg, at least about150 μg, at least about 200 μg, at least about 250 μg, at least about 300μg, at least about 350 μg, at least about 400 μg, at least about 450 μg,at least about 500 μg, at least about 550 μg, at least about 600 μg, atleast about 650 μg, at least about 700 μg, at least about 750 μg, atleast about 800 μg, at least about 850 μg, at least about 950 μg, or upto about 1000 μg of antigen. In embodiments where the antigen is arecombinant protein or peptide, a suitable dose can be about 10-100 μg.In embodiments where the antigen is a recombinant protein or peptide, asuitable dose can be about 10-100 μg.

Methods of Decreasing Recruitment of Monocytes to a Lymph Node

Vaccination can trigger the mobilization and recruitment of antigenpresenting cells (APC) to lymph nodes, e.g., vaccine draining lymphnodes. Monocytes, one subtype of APC, have recently been shown to have asuppressive effect on B cell and T cell proliferation, thus limitingimmune responses during vaccination. The present invention is based inpart on the observation that the administration of an effective amountof an ARB or a compound of Formula (I) (e.g., Ondansetron and/orAlosetron) in conjunction with an antigen decreases the recruitment ofmonocytes, e.g., CD14^(hi)CD16⁻ human monocytes, to a lymph node.

Accordingly, in one embodiment, the present invention includes a methodof decreasing the recruitment of monocytes to a lymph node in anindividual, comprising administering to the individual an antigen and anARB. In another embodiment, the present invention includes a method ofdecreasing the recruitment of monocytes to a lymph node in anindividual, comprising administering to the individual an antigen and acompound of Formula (I). In certain embodiments, the ARB or compound offormula (I) are administered in an effective amount, e.g., an amountsufficient to decreasing the recruitment of monocytes to a lymph node.In particular embodiments, the antigen, the ARB and/or the compound ofFormula (I) are present in one or more composition, e.g., one or morepharmaceutical compositions.

In certain embodiments, the methods of decreasing recruitment ofmonocytes to a lymph node provided by the invention includeadministering an effective amount of an ARB or a compound of Formula (I)(e.g., Ondansetron and/or Alosetron) to an individual in conjunctionwith an antigen. An individual to whom an ARB or a compound of Formula(I) (e.g., Ondansetron and/or Alosetron) is administered in conjunctionwith an antigen can exhibit a decrease in the number of monocytes thathave migrated to lymph node. In certain embodiments, the decrease in thenumber of monocytes can be at least about 5% less, at least about 10%less, at least about 15% less, at least about 20% less, at least about25% less, at least about 30% less, at least about 35% less, at leastabout 40% less, at least about 45% less, at least about 50% less, ormore than about 50% less (e.g., about 55%, about 60% or about 65% less)relative to the number of monocytes that have migrated to a lymph nodeof an individual that has received the antigen alone, including anyrange between about 5% less and about 65% less. In certain embodiments,the number of monocytes that have migrated to a lymph node in anindividual to whom an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) is administered in conjunction with anantigen can be more than 10% less, more than 20% less, more than 30%less, more than 40% less, more than 50% less, more than 60% less, morethan about 65% less, more than about 70% less, or more than about 75%less, more than 80% less, more than about 81% more than about 82% less,more than 83% less, more than 84% less, more than 85% less, more than86% less, more than 87% less, more than 88% less, more than 89% less,more than 90% less, at least about 91% less, at least about 93% less, orat least about 95% less than the number of monocytes that have migratedto a lymph node of an individual that has received the antigen alone,including any range between about 10% less and about 95% less.

In certain embodiments, the compound of Formula (I) (e.g., Ondansetronand/or Alosetron) administered in conjunction with the antigen can beany compound, combination of compounds, or any prodrug, salt, orderivative of a compound described herein. In certain embodiments, thecombination of compound of Formula (I) and antigen is any of thosedescribed herein. In some embodiments, Ondansetron can be administeredin conjunction with the antigen. In some embodiments, Alosetron can beadministered in conjunction with the antigen. The antigen administeredin conjunction with the compound of Formula (I) can be any antigendescribed herein. The antigen administered in conjunction with thecompound of Formula (I) can be provided in any amount described herein.The compound of Formula (I) and the antigen can be present in a singlepharmaceutical composition, or they can be provided in separatecompositions that can be administered in any order relative to oneanother or administered simultaneously, as described herein.Compositions containing the compound of Formula (I) and/or the antigencan be administered according to any method known in the art at dosagesdescribed elsewhere herein.

In certain embodiments, the individuals to whom a compound of Formula(I) is administered in conjunction with the antigen can be those who areotherwise not receiving Ondansetron or Alosetron for the treatment of,e.g., irritable bowel syndrome (IBS), post-operative nausea and vomiting(PONV), chemotherapy-induced nausea and vomiting (CINV),radiation-induced nausea and vomiting (RINV), and/or other conditions,as described herein. In some embodiments, an individual who has acondition or disease described herein, or is otherwise in need oftreatment, can receive Ondansetron and/or Alosetron in conjunction withan antigen for the purpose of enhancing an immune response if theindividual is not being treated with Ondansetron and/or Alosetron.Alternatively, in certain embodiments, the individuals who are beingtreated with Ondansetron and/or Alosetron can temporarily suspendOndansetron and/or Alosetron treatment. For example, an individual whois being treated with Ondansetron and/or Alosetron for a pre-existingcondition can receive Ondansetron and/or Alosetron conjunction with anantigen if the individual has taken Ondansetron and/or Alosetron fortreatment of the pre-existing condition more than about 10 minutes, morethan about 30 minutes, more than about 1 hour, more than about 3 hours,more than about 6 hours, more than about 12 hours, more than about 18hours, more than about 24 hours, more than about 1 day more than about 2days, more than about 3 days, more than about 4 days, more than about 5days, more than about 6 days, more than about 1 week, more than about 3weeks, more than about 1 month, more than about 2 months, more thanabout three months, more than about 4 months, more than about 5 months,more than about 6 months, more than about 7 months, more than about 8months, more than about 9 months, more than about 10 months, more thanabout 11 months, or more than about 1 year before receiving theOndansetron and/or Alosetron in conjunction with the antigen, includingany range in between these values. Alternatively, in other embodiments,the individuals to whom a compound of Formula (I) is administered inconjunction with an antigen can be those who have temporarily suspendedOndansetron and/or Alosetron treatment and have been shown, usingmethods well known in the art, to not have detectable levels ofOndansetron and/or Alosetron in their blood and/or urine prior to theadministration of Ondansetron and/or Alosetron in conjunction with theantigen.

In particular embodiments, the ARB administered in conjunction with theantigen can be any ARB, combination of ARBs, or any prodrug, salt, orderivative of an ARB described herein. The antigen administered inconjunction with the ARB can be any antigen described herein. In certainembodiments, the combination of the ARB and the antigen is any describedherein. The antigen administered in conjunction with the ARB can beprovided in any amount described herein. The ARB and the antigen can bepresent in a single pharmaceutical composition, or they can be providedin separate compositions that can be administered in any order relativeto one another or administered simultaneously, as described herein.

In particular embodiments, the individuals to whom an ARB isadministered in conjunction with an antigen can be those who areotherwise not receiving an ARB for treatment of a pre-existingcondition, e.g., hypertension, diabetic nephropathy, heart failure,and/or other conditions, as described herein. In some embodiments, anindividual who has a condition or disease described herein, or who isotherwise in need of treatment, can receive an ARB in conjunction withan antigen for the purpose of enhancing an immune response if theindividual is not being treated with an ARB. Alternatively, in someembodiments, the individuals to whom an ARB is administered inconjunction with an antigen can be those who have temporarily suspendedARB treatment. For example, an individual who is being treated with anARB for a pre-existing condition can receive an ARB in conjunction withan antigen if the individual has taken the ARB for treatment more thanabout 10 minutes, more than about 30 minutes, more than about 1 hour,more than about 3 hours, more than about 6 hours, more than about 12hours, more than about 18 hours, more than about 24 hours, more thanabout 1 day more than about 2 days, more than about 3 days, more thanabout 4 days, more than about 5 days, more than about 6 days, more thanabout 1 week, more than about 3 weeks, more than about 1 month, morethan about 2 months, more than about three months, more than about 4months, more than about 5 months, more than about 6 months, more thanabout 7 months, more than about 8 months, more than about 9 months, morethan about 10 months, more than about 11 months, or more than about 1year before receiving the ARB in conjunction with the antigen, includingany range in between these values. Alternatively, in certainembodiments, an individual who is being treated with an ARB for apre-existing condition can suspend ARB treatment and be tested, usingmethods well known in the art, to determine that the individual does nothave a detectable level of an ARB in their blood and/or urine prior tothe administration of the ARB in conjunction with the antigen.

Monocytes

Monocytes are agranular leukocytes that originate in the bone marrow andare released to the peripheral circulation as non-dividing cells.Monocytes constitute approximately 10% of peripheral leukocytes inhumans and approximately 4% of leukocytes in mice. Monocytes areequipped with chemokine receptors and adhesion receptors that mediatemigration from blood to tissues during infection, where they engulfpathogens and produce immune effector molecules. They can alsodifferentiate into inflammatory dendritic cells or macrophages duringinflammation.

In mice, circulating monocytes can be classified into two distinctpopulations, inflammatory monocytes and resident monocytes, based on theexpression of specific cell surface markers. Murine inflammatorymonocytes are categorized as CCR2⁺, CX3CR1^(low), and GR1⁺ (also knownas Ly6). The monocyte cell population found to suppress vaccine immunityprimarily includes CCR2 monocytes Mitchell et al. (2012) J. Immunology189: 5612-5621. Human monocyte subpopulations have also been identifiedbased on the differential expression of the antigenic markers CD16 andCD14. The human monocytes that correspond to murine inflammatorymonocytes are categorized as CD14^(high)CD16⁻.

Detecting a Reduction of Monocyte Recruitment to a Lymph Node

Vaccines can induce inflammation, which typically results in therecruitment of monocytes to the site of vaccination and to the lymphnodes, i.e., lymph nodes that lie immediately downstream of thevaccination site, or “vaccine draining lymph nodes.” Decreased monocytemigration to a lymph node can be demonstrated by showing that the numberof monocytes in, e.g., a vaccine draining lymph node of a humanindividual who received an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) in conjunction with the antigen, is lowerthan the number of monocytes in, e.g., a vaccine draining lymph node ofa human individual who received only the antigen. This can be assayed byperforming flow cytometry on tissue obtained from each individual'slymph nodes and, e.g., determining whether fewer CD14^(hi)CD16⁻monocytes are detected in the tissue from the individual who receivedthe ARB or the compound of Formula (I) (e.g., Ondansetron and/orAlosetron) in conjunction with the antigen than in the tissue from anindividual who did not receive the ARB or the compound of Formula (I).In certain embodiments, decreased monocyte migration to a lymph node,e.g., a vaccine draining lymph node, can be demonstrated by detecting achange in the circulating levels of, e.g., total monocytes orinflammatory monocyte, within the first 24 hours after the ARB or thecompound of Formula (I) is administered to the individual in conjunctionwith the antigen.

Methods of determining the amount of circulating CD14^(hi)CD16⁻monocytes or circulating inflammatory monocytes are known in the art.For example, blood specimens can be collected from individuals andprepared for flow cytometry using labeled antibodies againstCD14^(hi)CD16⁻ monocyte-specific cell surface markers or againstinflammatory monocyte cell surface markers, such as those describedelsewhere herein. Absolute numbers of monocytes can be calculated usingleukocyte counts derived from an automated blood cell counter.Alternatively, skin biopsies taken at or near, e.g., vaccine injectionsites, can be collected from individuals within 24 hours of receiving avaccine in conjunction with an ARB, and the biopsied tissue can preparedso that the number of monocytes in the tissue can be quantified andcompared to the number of monocytes counted in a tissue obtained from anindividual who received the vaccine alone. For example, an aspirate ofthe draining lymph node, e.g., nearest to the vaccine injection site,can be taken via ultrasound imaging, and the number of monocytes in theaspirate can be quantified and compared between an individual whoreceived an ARB in conjunction with a vaccine and an individual whoreceived the vaccine alone. Additional methods of determining levels ofcirculating monocytes that can be used are described in, e.g., Aldonyteet al. (2003) Resp. Res. 4:11; Janciauskiene et al. (2001)Atherosclerosis 158: 41-51; Nockher et al. (1998) Infect Immun. 66:2782-2790; and others.

Methods of Amplifying Vaccine Immunity

Vaccines have played a key role in reducing the incidence ofdebilitating and/or fatal diseases. Vaccine immunity depends on theability of individuals to mount a robust immune response. As notedabove, inflammatory monocytes are rapidly recruited to the site ofvaccination and to lymph nodes, where they suppress B cell and T cellresponses. This a particular public health concern in, e.g., vulnerablepopulations that exhibit reduced immune responsiveness, such as theyoung, the elderly, and the immunocompromised. The invention providesmethods that can be beneficially used to substantially amplify vaccineimmunity. As used herein, “amplifying vaccine immunity” refers toincreasing a vaccine-induced immune response, e.g., a vaccine-inducedhumoral (or B-cell) immune response and/or a vaccine-induced cellular(or T-cell) immune response.

Accordingly, in one embodiment, the present invention includes a methodof amplifying vaccine immunity in an individual, comprisingadministering to the individual a vaccine and an ARB. In anotherembodiment, the present invention includes a method of amplifyingvaccine immunity in an individual, comprising administering to theindividual a vaccine and a compound of Formula (I). In certainembodiments, the ARB or compound of formula (I) are administered in aneffective amount, e.g., an amount sufficient to amplify vaccineimmunity. In particular embodiments, the antigen, the ARB and/or thecompound of Formula (I) are present in one or more composition, e.g.,one or more pharmaceutical compositions.

In certain embodiments, the methods include administering an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) to anindividual in conjunction with a vaccine. An amplified humoral (orB-cell) immune response can be demonstrated by showing that the antibodytiters from, e.g., an individual who received an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) in conjunction with thevaccine, are higher than antibody titers from, e.g., an individual whoreceived only the vaccine. In certain embodiments, an individual to whoman ARB or a compound of Formula (I) (e.g., Ondansetron and/or Alosetron)was administered in conjunction with a vaccine or an antigen can exhibita humoral immune response that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 20-fold, at least about50-fold, at least about 60-fold, at least about 70-fold, at least about80-fold, at least about 90 fold, at least about 100-fold, at least about110-fold, at least about 125-fold, at least about 150-fold, at leastabout 175-fold, at least about 200-fold, or more than 200-fold (e.g.,about 250-fold, about 300-fold, or about 350-fold) higher than thehumoral immune response exhibited by an individual to whom vaccine alonewas administered, including any range in between about 2-fold and about300-fold. In certain embodiments, an individual to whom an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) wasadministered in conjunction with a vaccine or an antigen can exhibit acellular immune response that is at least about 1.2-fold, at least about1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about3-fold, at least about 3.5 fold, at least about 4-fold, at least about4.5-fold, at least about 5-fold, or more than about 5-fold (e.g., about5.5-fold, about 6-fold, or about 6.5-fold) higher than the cellularimmune response exhibited by an individual to whom vaccine alone wasadministered, including any range between about 1.2-fold and about 6.5fold.

In particular embodiments, the compound of Formula (I) administered inconjunction with the vaccine can be any compound, combination ofcompounds, or any prodrug, salt, or derivative of a compound describedherein. In particular embodiments, the vaccine is any described herein.In particular embodiments, the combination of vaccine and compound ofFormula (I) is any described herein. In some embodiments, the vaccinecan be administered in conjunction with Ondansetron. In someembodiments, the vaccine can be administered in conjunction withAlosetron. The compound of Formula (I) and the vaccine can be present ina single pharmaceutical composition, or they can be provided in separatecompositions that can be administered in any order relative to oneanother or administered simultaneously, as described herein.Compositions containing the compound of Formula (I) and/or the vaccinecan be administered according to any method known in the art at dosagesdescribed elsewhere herein.

In particular embodiments, the ARB administered in conjunction with thevaccine can be any ARB, combination of ARBs, or any prodrug, salt, orderivative of an ARB described herein. In particular embodiments, thevaccine is any described herein. In particular embodiments, thecombination of vaccine and ARB is any described herein. The ARB andvaccine can be present in a single pharmaceutical composition, or theycan be provided in separate compositions that can be administered in anyorder relative to one another or administered simultaneously, asdescribed herein. Compositions containing the ARB and/or the vaccine canbe administered according to any method known in the art at dosagesdescribed elsewhere herein.

In particular embodiments, the individuals to whom an a compound ofFormula (I) is administered in conjunction with the vaccine can be thosewho are otherwise not receiving Ondansetron or Alosetron for thetreatment of, e.g., irritable bowel syndrome (IBS), post-operativenausea and vomiting (PONV), chemotherapy-induced nausea and vomiting(CINV), radiation-induced nausea and vomiting (RINV), and/or otherconditions, as described herein. In some embodiments, an individual whohas a condition or disease described herein, or is otherwise in need oftreatment, can receive Ondansetron and/or Alosetron in conjunction witha vaccine for the purpose of enhancing an immune response if theindividual is not being treated with Ondansetron and/or Alosetron.Alternatively, in some embodiments, the individuals on whom the methodsare practiced have temporarily suspended Ondansetron and/or Alosetrontreatment and have been shown, using methods well known in the art, tonot have detectable levels of Ondansetron and/or Alosetron in theirblood and/or urine prior to the administration of the Ondansetron and/orAlosetron in conjunction with the vaccine.

In particular embodiments, the individuals to whom an ARB isadministered in conjunction with a vaccine can be those who areotherwise not receiving an ARB for treatment of a pre-existingcondition, e.g., hypertension, diabetic nephropathy, heart failure,and/or other conditions, as described herein. In another embodiment, anindividual who has a condition or disease described herein, or who isotherwise in need of treatment, can receive an ARB in conjunction with avaccine for the purpose of enhancing an immune response if theindividual is not being treated with an ARB. Alternatively, in someembodiments, the individuals to whom an ARB is administered inconjunction with a vaccine can be those who have temporarily suspendedARB treatment. For example, an individual who is being treated with anARB for a pre-existing condition can receive an ARB in conjunction witha vaccine if the individual has taken the ARB for treatment more thanabout 10 minutes, more than about 30 minutes, more than about 1 hour,more than about 3 hours, more than about 6 hours, more than about 12hours, more than about 18 hours, more than about 24 hours, more thanabout 1 day more than about 2 days, more than about 3 days, more thanabout 4 days, more than about 5 days, more than about 6 days, more thanabout 1 week, more than about 3 weeks, more than about 1 month, morethan about 2 months, more than about three months, more than about 4months, more than about 5 months, more than about 6 months, more thanabout 7 months, more than about 8 months, more than about 9 months, morethan about 10 months, more than about 11 months, or more than about 1year before receiving the ARB in conjunction with the vaccine, includingany range in between these values. Alternatively, in some embodiments,an individual who is being treated with an ARB for a pre-existingcondition can suspend ARB treatment and be tested, using methods wellknown in the art, to determine that the individual does not have adetectable level of an ARB in their blood and/or urine prior to theadministration of the ARB in conjunction with the vaccine.

Vaccines

A vaccine is a composition that contains an antigen which, whenadministered to an individual, stimulates an immune response. Vaccinesconfer long-term immunity by inducing the development of immune memorycells that are able to mount a strong response if the pathogen isdetected again. The vaccine that is administered in the methods can beany vaccine or vaccine formulation known to those of skill in the art.For example, the vaccine administered in conjunction with an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) can containany antigen known in the art. In certain embodiments, the vaccineadministered in conjunction with an ARB or a compound of Formula (I)(e.g., Ondansetron and/or Alosetron) can contain any antigen orcombination of antigens described herein, e.g., a live infectious agent,a killed infectious agent, a polysaccharide, or a toxin produced by aninfectious agent. In certain embodiments of the methods, the vaccinecontains specific proteins, e.g., purified from an infection agent orrecombinantly produced. In certain embodiments of the methods, thepolysaccharide or protein present in the vaccine is conjugated to animmuno-stimulating molecule, such as a carrier protein.

In some embodiments, the vaccine administered in conjunction with theARB or the compound of Formula (I) (e.g., Ondansetron and/or Alosetron)can be a DNA vaccine, which entails direct introduction into appropriatetissues of, e.g., a plasmid encoding the antigen(s) against which animmune response is sought, and which relies on the in situ production ofthe target antigen. Further details regarding DNA vaccines are describedin, e.g., DNA Vaccines: Methods and Protocols (Douglas B. Lourie andRobert Whalen, eds., 2000) Humana Press; DNA Vaccines (Mark Saltzman,Hong Shen, and Janet Brandsma, eds. 2006) Human Press; and others.

In certain embodiments, the vaccine administered in conjunction with anARB or a compound of Formula (I) (e.g., Ondansetron and/or Alosetron)can be a viral vector vaccine, which is typically a live attenuatedvirus that is genetically engineered to carry DNA encoding proteinantigens from an unrelated organism. Viral vector vaccines carry DNAinto a host cell for production of antigenic proteins that can betailored to stimulate an immune responses. Viral vector vaccines, unlikeDNA vaccines, also have the potential to actively invade host cells andreplicate, much like a live attenuated vaccine, further activating theimmune system like an adjuvant. Further details regarding viral vectorvaccines are described in, I, Bråve et al. (2007) Mol Pharm. 4: 18-32;Kaufmann et al. (2012) Trends Mol Med. 18: 365-7; Ulmer et al. (2012)Vaccine. 30: 4414-4418; and others.

In certain embodiments, the vaccine administered in conjunction with anARB or a compound of Formula (I) (e.g., Ondansetron and/or Alosetron)can be a dendritic cell vaccine. Dendritic cells (DCs) areantigen-presenting cells that are involved in the induction of primaryimmune responses. The unique ability of DCs to activate naive and memoryCD4⁺ and CD8⁺ T cells suggests that they could be used, e.g., for theinduction of a specific anti-tumor immunity. An individual's DCs can beharvested, pulsed with one or more antigens, and used to re-immunize theindividual to induce a protective immune response. Further detailsregarding dendritic cell vaccines are described in, e.g., Pellegatta etal. (2009) Methods Mol Biol. 568: 233-247; Bhargava et al. (2012)Immunotherapy. 4: 703-718; Van Brussel et al. (2012) Mediators Inflamm.Article ID 690643; Yamanaka et al. (2012) Adv Exp Med Biol. 746:187-200;and others.

Methods of Inhibiting Tumor Growth or Metastasis

Therapeutic cancer vaccines stimulate the immune system to recognizetumor-specific antigens and to generate an immune response to find anddestroy cells that express them.

Producing effective therapeutic vaccines has been difficult, asgenetically unstable cancer cells are capable of evading immunerecognition. Recent evidence indicates that monocyte depletion canincrease the efficacy of cancer vaccine (Mitchell et al. (2012)“Suppression of Vaccine Immunity by Inflammatory Monocytes.” J.Immunology 189: 5612-5621 and U.S. Patent Application Publication No. US2012/0156280). The angiotensin II receptor blockers (ARB) and thecompounds of Formula (I) described herein (e.g., Ondansetron orAlosetron) can be used to inhibit monocyte migration, thus amplifyingcancer vaccine immunity by enhancing an immune response.

Accordingly, in one embodiment, the present invention includes a methodof inhibiting tumor growth or metastasis in an individual, comprisingadministering to the individual an anti-tumor preparation and an ARB. Inanother embodiment, the present invention includes a method ofinhibiting tumor growth or metastasis in an individual, comprisingadministering to the individual an anti-tumor preparation and a compoundof Formula (I). In particular embodiments, the anti-tumor preparation isany described herein. In particular embodiments, the combination of ananti-tumor preparation and a compound of Formula (I) is any describedherein. In certain embodiments, the ARB or compound of formula (I) areadministered in an effective amount, e.g., an amount sufficient toinhibit tumor growth or metastasis (e.g., when administered with ananti-tumor preparation). In particular embodiments, the antigen, the ARBand/or the compound of Formula (I) are present in one or morecomposition, e.g., one or more pharmaceutical compositions. Inparticular embodiments, the anti-tumor preparation and the ARB orcompound of Formula (I) are present in a single composition. Inparticular embodiments, the individual has a tumor.

In particular embodiments, these uses/methods include administering aneffective amount of an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) in conjunction with an anti-tumorpreparation to an individual. In some embodiments, the ARB or thecompound of Formula (I) (e.g., Ondansetron and/or Alosetron) isadministered in conjunction with an anti-tumor preparation to inhibitthe tumor growth or metastasis of an epithelial cancer, breast cancer,prostate cancer, colon cancer, a hematopoietic cancer, leukemia,lymphoma, a sarcoma, melanoma, a head sarcoma, a neck sarcoma, asquamous cell carcinoma, an osteosarcoma, or a brain tumor.

In some embodiments, tumor growth in an individual to whom an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) isadministered in conjunction with an anti-tumor preparation is reduced byat least about 5%, at least about 10%, at least about 20%, at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, or at least about 95%as compared to tumor growth in an individual who received the anti-tumorpreparation alone. In some embodiments, tumor metastasis in anindividual to whom an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) is administered in conjunction with ananti-tumor preparation is reduced by at least about 5%, at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, or at least about 95% as compared to tumor metastasisin an individual who received the anti-tumor preparation alone.

Tumor metastasis can be measured by any suitable method known to theskilled artisan. In certain embodiments, tumor metastases are measuredby counting the number of tumor nodules (i.e., the number of tumormetastases present in an individual). In certain embodiments, tumormetastases are measured by determining the overall size of the tumornodules, or the volume of tumor nodules as measured by CT or MRI.

In certain embodiments, the compound of Formula (I) (e.g., Ondansetronand/or Alosetron) administered in conjunction with the anti-tumorpreparation can be any compound, combination of compounds, or anyprodrug, salt, or derivative of compound of Formula (I) herein. In someembodiments, the anti-tumor preparation can be administered inconjunction with Alosetron. In some embodiments, the anti-tumorpreparation can be administered in conjunction with Ondansetron. Thecompound of Formula (I) and an anti-tumor preparation can be present ina single pharmaceutical composition, or they can be provided in separatecompositions that can be administered in any order relative to oneanother or administered simultaneously, as described herein.Compositions containing the compound of Formula (I) (e.g., Ondansetronand/or Alosetron) and/or the anti-tumor preparation can be administeredaccording to any method known in the art at dosages described elsewhereherein.

In certain embodiments, the individuals to whom a compound of Formula(I) is administered in conjunction with the anti-tumor preparation canbe those who are otherwise not receiving Ondansetron or Alosetron forthe treatment of, e.g., irritable bowel syndrome (IBS), post-operativenausea and vomiting (PONV), chemotherapy-induced nausea and vomiting(CINV), radiation-induced nausea and vomiting (RINV), and/or otherconditions, as described herein. In some embodiments, an individual whohas a condition or disease described herein, or is otherwise in need oftreatment, can receive Ondansetron and/or Alosetron in conjunction withan anti-tumor preparation for the purpose of enhancing an immuneresponse if the individual is not being treated with Ondansetron and/orAlosetron. Alternatively, in some embodiments, the individuals on whomthe methods are practiced have temporarily suspended Ondansetron and/orAlosetron treatment and have been shown, using methods well known in theart, to not have detectable levels of Ondansetron and/or Alosetron intheir blood and/or urine prior to the administration of the Ondansetronand/or Alosetron in conjunction with the anti-tumor preparation.

In particular embodiments, the ARB administered in conjunction with theanti-tumor preparation can be any ARB, combination of ARBs, or anyprodrug, salt, or derivative of an ARB described herein. The ARB andanti-tumor preparation can be present in a single pharmaceuticalcomposition, or they can be provided in separate compositions that canbe administered in any order relative to one another or administeredsimultaneously, as described herein. Compositions containing the ARBand/or the anti-tumor preparation can be administered according to anymethod known in the art at dosages described elsewhere herein.

In certain embodiments, the individuals to whom an ARB is administeredin conjunction with an anti-tumor preparation can be those who areotherwise not receiving an ARB for treatment of a pre-existingcondition, e.g., hypertension, diabetic nephropathy, heart failure,and/or other conditions, as described herein. In some embodiments, anindividual who has a condition or disease described herein, or who isotherwise in need of treatment, can receive an ARB in conjunction withan anti-tumor preparation for the purpose of enhancing an immuneresponse if the individual is not being treated with an ARB.Alternatively, in some embodiments, the individuals to whom an ARB isadministered in conjunction with an anti-tumor preparation can be thosewho have temporarily suspended ARB treatment. For example, an individualwho is being treated with an ARB for a pre-existing condition canreceive an ARB in conjunction with an anti-tumor preparation if theindividual has taken the ARB for treatment more than about 10 minutes,more than about 30 minutes, more than about 1 hour, more than about 3hours, more than about 6 hours, more than about 12 hours, more thanabout 18 hours, more than about 24 hours, more than about 1 day morethan about 2 days, more than about 3 days, more than about 4 days, morethan about 5 days, more than about 6 days, more than about 1 week, morethan about 3 weeks, more than about 1 month, more than about 2 months,more than about three months, more than about 4 months, more than about5 months, more than about 6 months, more than about 7 months, more thanabout 8 months, more than about 9 months, more than about 10 months,more than about 11 months, or more than about 1 year before receivingthe ARB in conjunction with the anti-tumor preparation, including anyrange in between these values. Alternatively, in some embodiments, anindividual who is being treated with an ARB for a pre-existing conditioncan suspend ARB treatment and be tested, using methods well known in theart, to determine that the individual does not have a detectable levelof an ARB in their blood and/or urine prior to the administration of theARB in conjunction with the anti-tumor preparation.

Anti-Tumor Preparations

The anti-tumor preparation administered in conjunction with the ARB orthe compound of Formula (I) (e.g., Ondansetron and/or Alosetron) can beany anti-tumor preparation known and used in the art. For example,certain anti-tumor preparations inhibit the synthesis of new DNAstrands, thus preventing tumor cells from replicating. In certainembodiments, the anti-tumor preparation administered in conjunction withthe ARB or the compound of Formula (I) (e.g., Ondansetron and/orAlosetron) can be an antimetabolite, such as 5-fluorouracil,methotrexate, capecitabine, Alimta, gemcitabine, etc. In certainembodiments, the anti-tumor preparation can be a platinum-based agent,such as cisplatin, carboplatin, oxaliplatin, and the like, whichcross-link DNA and inhibit DNA repair in tumor cells. In certainembodiments, the anti-tumor preparation can be an alkylating agent, suchas cyclophosphamide, carmustine (BCNU), methyl-CCNU, or piposulfan. Incertain embodiments, the anti-tumor agent can be a tyrosine kinaseinhibitor, such as gefitinib (Iressa®), imatinib (Gleevec®), lapatinib,sunitinib (Sutent®), or Tarceva. In certain embodiments, the anti-tumorpreparation can be an anthracycline, such as actinomycin, doxil,doxorubicin (adriamycin), epirubicin, or mitoxantrone. In certainembodiments, the anti-tumor preparation can be a topoisomeraseinhibitor, such as camptothecin, irinotecan, topotecan, etoposide,amsacrine, etoposide phosphate, or teniposide.

Other anti-tumor preparations interfere with microtubule assembly ordisassembly, thus interrupting tumor cell division. In certainembodiments, the anti-tumor preparation can be a vinca alkaloid, such asvinblastine, vincristine, vindesine, VP-16, or vinorelbine (Navelbine®).Other anti-tumor agents that exhibit anti-microtubule activity includecolchicine, taxanes and taxane derivatives. Additional anti-tumorpreparations that can be administered in conjunction with an ARB or acompound of Formula (I) include proteasome inhibitors (e.g., bortezomib(Velcade)); anti-angiogenesis agents; and therapeutic antibodies (e.g.,anti-VEGF antibody/Avastin®/bevacizumab, anti-HER2antibody/Herceptin®/trastuzumab, Erbitux®/cetuximab,Campath/Alemtuzumab, Myelotarg/gemtuzumab, Zevalin/ibritumomab tiuextan,Rituxan/rituximab, and Bexxar/tositumomab). In certain embodiments, twoor more anti-tumor preparations can be administered in conjunction withan ARB or a compound of Formula (I) (e.g., Ondansetron and/orAlosetron).

In certain embodiments, the anti-tumor preparation that is administeredin conjunction with an ARB or a compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) can be a cancer vaccine, e.g., a cancervaccine designed to increase the targeted immune response against cancercells already present in the individual. In certain embodiments, thecancer vaccine administered in conjunction with an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) can contain wholeinactivated tumor cells, parts of tumor cells, or tumor cell lysates. Incertain embodiments, the cancer vaccine includes a tumor-specificantigen, such as a protein, peptide, or carbohydrate. In certainembodiments, the cancer vaccine that is administered in conjunction withan ARB or a compound of Formula (I) (e.g., Ondansetron and/or Alosetron)can include a nucleic acid, viral vector, or bacterial vector thatencodes a tumor-specific antigen. In certain embodiments, the cancervaccine that is administered in conjunction with an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) can be a dendritic cellvaccine that has been pulsed with a tumor-specific antigen. Thetumor-specific antigen can be any tumor-specific antigen known in theart, including, but not limited to, e.g., melanA/MART-, NY-ESO-1, MAGE,ETA, CEA, CA-125, CA15-3, CA27-29, CA19-9, MUC-1, AFP, an abnormalproduct of the ras gene, an abnormal product of the p53 gene, CAMEL,Ep-CAM, her-2/neu, WT-1, EBNA3, CD10, CD34, CD99, CD117, CD45 (PTPRC),chromogranin, mucin, LMP2, E6, E7, K12, K8.1, tyrosinase, proteinase 3,GM2, GD2, GD3, polysialic acid, fucosyl GM1, globo H, KSA, sialylLe^(a), Le^(y), TF, Tn, sTn, PSMA, PSA, MUC16, SAGE1, HBA-71,calretinin, carcinoembryonic antigen, a cytokeratin, desmin, EMA, FactorVIII, CD31 FL1, GFAP, GCDFP-15, HMB-25, an immunoglobulin, inhibin,keratin, a lymphocyte marker, Myo D1, MSA, neuofilament, NSE, PLAP,S100, SMA, synaptophysin, thyroglobulin, vimentin, tumor M2-PK, thyroidtranscription factor-1, a squamous cell carcinoma tumor-specificantigen, an osteosarcoma tumor-specific antigen, or a braintumor-specific antigen. Alternatively, the cancer vaccine can include apeptide or polysaccharide derived from any of the tumor-specific antigenlisted above.

Administering an ARB or a Compound of Formula (I) in Conjunction with anAntigen, Vaccine, or Anti-Tumor Preparation

Various methods described above entail administering an ARB or acompound of Formula (I) (e.g., Ondansetron and/or Alosetron) inconjunction with an antigen, a vaccine, or an anti-tumor preparation.The ARB or the compound of Formula (I) (e.g., Ondansetron and/orAlosetron) and the antigen, vaccine, or anti-tumor preparation can bepresent in a single composition. Alternatively, the ARB or the compoundof Formula (I) (e.g., Ondansetron and/or Alosetron) and the antigen,vaccine, or anti-tumor preparation can be provided in separatecompositions. For example, the ARB or the compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) can be provided as a tablet for oraladministration, and the antigen, vaccine, or anti-tumor preparation canbe provided in an injectable composition. Where the ARB or the compoundof Formula (I) (e.g., Ondansetron and/or Alosetron) and the antigen,vaccine, or anti-tumor preparations are present in separatecompositions, the two compositions can be administered in any orderrelative to one another. For example, the ARB or the compound of Formula(I) (e.g., Ondansetron and/or Alosetron) can be administered before theantigen, vaccine, or anti-tumor preparation. Alternatively, the antigen,vaccine, or anti-tumor preparation can be administered before the ARB orthe compound of Formula (I) (e.g., Ondansetron and/or Alosetron). Thepharmaceutical composition containing the ARB or the compound of Formula(I) (e.g., Ondansetron and/or Alosetron) and the pharmaceuticalcomposition containing the antigen, vaccine, or anti-tumor preparationcan be administered within at least about 12 hours, within at leastabout 11 hours, within at least about 10 hours, within at least about 9hours, within at least about 8 hours, within at least about 7 hours,within at least about 6 hours, within at least about 5 hours, within atleast about 4 hours, within at least about 3 hours, within at leastabout 2 hours, within at least about 1 hour, or less than 1 hour (e.g.,within about 45 minutes, within about 30 minutes, within about 15minutes, within about 10 minutes, within about 5 minutes, less than 5minutes, less than 4 minutes, less than 3 minutes, less than 2 minutes,or less than 1 minute) of one another, including any ranges betweenthese values. In certain embodiments, the two compositions can beadministered simultaneously, e.g., premixed and administered.

Accordingly, in certain embodiments, the compositions of the inventioninclude Ondansetron and an antigen, vaccine, or anti-tumor preparation.In certain embodiments, the composition is administered orally. Incertain embodiments, Ondansetron in the orally administered compositionis at a concentration sufficient to provide a dose of at least about 0.5mg, at least about 1 mg, at least about 2 mg, at least about 3 mg, atleast about 4 mg, at least about 5 mg, at least about 6 mg, at leastabout 7 mg, at least about 8 mg, at least about 10 mg, or less thanabout 12 mg Ondansetron, including any range in between these values. Incertain embodiments, the Ondansetron in the orally administeredcomposition is at a concentration sufficient to provide a dose of morethan about 24 mg, more than about 26 mg, more than about 28 mg, morethan about 30 mg, more than about 32 mg, or more than about 34 mgOndansetron, including any range in between these values.

In certain embodiments, compositions are administered intravenously.Compositions for intravenous administration can include Ondansetron at aconcentration sufficient to provide a dose of at least about 0.01mg/kg/day, at least about 0.05 mg/kg/day, at least about 0.1 mg/kg/day,at least about 0.15 mg/kg/day, at least about 0.2 mg/kg/day, at leastabout 0.3 mg/kg/day, at least about 0.4 mg/kg/day, at least about 0.5mg/kg/day, at least about 0.75 mg/kg/day, at least about 1.0 mg/kg/day,at least about 1.5 mg/kg/day, at least about 2.0 mg/kg/day, at leastabout 2.5 mg/kg/day, at least about 3.0 mg/kg/day, at least about 3.5mg/kg/day, at least about 4.0 mg/kg/day, at least about 4.5 mg/kg/day,at least about 5.0 mg/kg/day, at least about 5.5 mg/kg/day, at leastabout 6.0 mg/kg/day, at least about 6.5 mg/kg/day, at least about 7.0mg/kg/day, at least about 7.5 mg/kg/day, at least about 8.0 mg/kg/day,or less than about 8.5 mg/kg/day of Ondansetron, including any rangebetween these values.

For example, the invention provides compositions that can includeAlosetron and an antigen, vaccine, or anti-tumor preparation, where theAlosetron in the composition is at a concentration sufficient to providea dose of at least about 0.1 mg, at least about 0.2 mg, at least about0.3 mg, at least about 0.4 mg, or less than about 0.5 mg Alosetron,including any range between these values. In certain embodiments, theAlosetron in the composition is at a concentration sufficient to providea dose of more than about 2 mg, more than about 3 mg, more than about 4mg, more than about 5 mg, or more than about 6 mg Alosetron, includingany range in between these values.

In certain embodiments, the ARB administered in conjunction with theantigen, vaccine, or anti-tumor preparation can be any ARB, combinationof ARBs, or any prodrug, salt, or derivative of an ARB described herein.For example, Losartan can be administered in conjunction with theantigen, vaccine, or anti-tumor preparation at a concentrationsufficient to provide a dose of at least about 5 mg, at least about 10mg, at least about 15 mg, at least about 20 mg, or less than about 25 mgof Losartan, including any range in between these values. In certainembodiments, the Losartan in the composition is at a concentrationsufficient to provide a dose of more than about 80 mg, more than about100 mg, more than about 125 mg, more than about 150 mg, more than about175 mg, or more than about 200 mg or Losartan, including any range inbetween about 5 mg and about 200 mg.

In certain embodiments, compositions of the invention can includeLosartan at a concentration sufficient to provide a dose of at leastabout 0.5 mg/kg, at least about 0.75 mg/kg, at least about 1.0 mg/kg, atleast about 1.25 mg·kg, at least about 1.5 mg/kg, at least about 1.75mg/kg, or at least about 2.0 mg/kg of Losartan, including any rangebetween about 0.5 mg/kg and about 1.75 mg/kg. In certain embodiments,compositions of the invention can include Losartan at a concentrationsufficient to provide a dose of more than about 1.75 mg/kg, at leastabout 2.0 mg/kg, at least about 5 mg/kg, at least about 7 mg/kg, atleast about 10 mg/kg, at least about 12 mg/kg, at least about 15 mg/kg,at least about 17 mg/kg, at least about 20 mg/kg, at least about 22mg/kg, at least about 25 mg/kg, at least about 27 mg/kg, or at leastabout 30 mg/kg or Losartan, including any range in between about 1.75mg/kg and about 30 mg/kg. In certain embodiments, the compositions ofthe invention can include Losartan at a concentration sufficient toprovide a dose of more than about 30 mg/kg, e.g., at least about 35mg/kg or at least about 40 mg/kg of Losartan, including any range inbetween about 30 mg/kg and about 40 mg/kg.

In certain embodiments, Candesartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 4 mg, less than about 3 mg, less than about 2 mg, or lessthan about 1 mg, including any range between these values. In certainembodiments, Candesartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 32 mg, more than about 40 mg, more than about 48 mg, more thanabout 56 mg, or more than about 64 mg, including any range in betweenthese values.

In certain embodiments, Erposartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 400 mg, less than about 300 mg, less than about 200 mg, lessthan about 100 mg, or less than about 50 mg, including any range betweenthese values. In certain embodiments, Eprosartan can be administered inconjunction with an antigen, vaccine, or anti-tumor preparation at adosage of more than about 600 mg, more than about 750 mg, more thanabout 900 mg, more than about 1050 mg, or more than about 1200 mg,including any range in between these values.

In certain embodiments, Irbesartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of aboutless than 150 mg, less than about 100 mg, less than about 50 mg, or lessthan about 25 mg, including any range between these values. In certainembodiments, Irbesartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 300 mg, more than about 375 mg, more than about 450 mg, more thanabout 525 mg, or more than about 600 mg, including any range in betweenthese values.

In certain embodiments, Olmesartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 20 mg, less than about 15 mg, less than about 10 mg, or lessthan about 5 mg, including any range between these values. In certainembodiments, Olmesartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 40 mg, more than about 50 mg, more than about 60 mg, more thanabout 70 mg, or more than about 40 mg, including any range in betweenthese values.

In certain embodiments, Telmisartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 20 mg, less than about 15 mg, less than about 10 mg, or lessthan about 5 mg, including any range between these values. In certainembodiments, Telmisartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 80 mg, more than about 100 mg, more than about 120 mg, more thanabout 140 mg, or more than about 160 mg, including any range in betweenthese values.

In certain embodiments, Valsartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 20 mg, less than about 15 mg, less than about 10 mg, or lessthan about 5 mg, including any range between these values. In certainembodiments, Valsartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 320 mg, more than about 400 mg, more than about 480 mg, more thanabout 560 mg, or more than about 640 mg, including any range in betweenthese values.

In certain embodiments, Azilsartan can be administered in conjunctionwith an antigen, vaccine, or anti-tumor preparation at a dosage of lessthan about 40 mg, less than about 30 mg, less than about 20 mg, or lessthan about 10 mg, including any range between these values. In certainembodiments, Azilsartan can be administered in conjunction with anantigen, vaccine, or anti-tumor preparation at a dosage of more thanabout 80 mg, more than about 100 mg, more than about 120 mg, more thanabout 140 mg, or more than about 160 mg, including any range in betweenthese values.

Modes of Administration

Administration of the ARB or the compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) in conjunction with an antigen, vaccine,or anti-tumor preparation can be achieved by a variety of routes,including, e.g., topical application, inhalation, intravenous injection,application to a wound site, application to a surgical site,intracavitary injection, by suppository, subcutaneously, intradermally,transcutaneously, by nebulization, intraplurally, intraventricularly,intra-articularly, intraocularly, intraspinally, or by other methodswell known to those of skill in the art. In certain embodiments, the ARBor the compound of Formula (I) (e.g., Ondansetron and/or Alosetron) andthe antigen, vaccine, or anti-tumor preparation can be systemic afteradministration or may be localized by the use of regionaladministration, intramural administration, or use of an implant thatacts to retain the active dose at the site of implantation. When the ARBor the compound of Formula (I) (e.g., Ondansetron and/or Alosetron) andthe antigen, vaccine, or anti-tumor preparation are present in twoseparate pharmaceutical compositions, each composition can beadministered through a different route or through the same route. Incertain embodiments, the ARB or the compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) can be administered in conjunction withthe antigen, vaccine, or anti-tumor preparation using any medicallyappropriate procedure, e.g. intravascular (intravenous, intraarterial,intracapillary) administration, injection into the cerebrospinal fluid,intracavity or direct injection in the brain. Intrathecal administrationmay be carried out through the use of an Ommaya reservoir, in accordancewith known techniques. (F. Balis et al., Am J. Pediatr. Hematol. Oncol.11, 74, 76 (1989). In certain embodiments, the ARB or the compound ofFormula (I) (e.g., Ondansetron and/or Alosetron) can be administered inconjunction with the antigen, vaccine, or anti-tumor preparation priorto exposure of the individual to, e.g., an infectious agent, so that theresulting immune response upon subsequent exposure to the infectiousagent can reduce the severity and/or duration of, e.g., the infection ordisease caused by the infectious agent. In certain embodiments, aneffective amount of the ARB or the compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) can be provided in one or moreadministrations. For example, where the antigen, vaccine, or anti-tumorpreparation and the ARB or the compound of Formula (I) (e.g.,Ondansetron and/or Alosetron) are provided in separate pharmaceuticalcompositions, an individual can receive the antigen, vaccine, oranti-tumor preparation and effective amount of an ARB or a compound ofFormula (I) (e.g., Ondansetron and/or Alosetron), and then receiveadditional administrations of the ARB or the compound of Formula (I)(e.g., Ondansetron and/or Alosetron) 12 hours, 24 hours, 36 hours, 48hours, and/or more than 48 hours after the initial administration.

For therapeutic treatment, in certain embodiments, the ARB or thecompound of Formula (I) (e.g., Ondansetron and/or Alosetron) can beadministered in conjunction with the antigen, vaccine, or anti-tumorpreparation to an individual who is already exposed to the pathogen orhas the infection or disease, e.g., cancer. The resulting enhancedimmune response can reduce the duration and/or severity of the existingdiseases or infection, as well as minimize any harmful consequences ofuntreated disease or infection. The ARB or the compound of Formula (I)(e.g., Ondansetron and/or Alosetron) can be administered in conjunctionwith the antigen, vaccine, or anti-tumor preparation along with anyother therapeutic regimen.

Individuals Who can be Treated Using the Methods

Various individuals can be treated using the methods described herein.In certain embodiments, the individual who can be treated using themethods described above can be a patient.

In certain embodiments, an ARB (e.g., Losartan) or a combination of ARBscan be administered in conjunction with the antigen, vaccine, oranti-tumor preparation to individuals who are otherwise not receiving anARB for treatment of a pre-existing condition. As noted elsewhereherein, ARBs can be prescribed for the treatment and/or prevention of avariety of diseases. Accordingly, it will be appreciated by one of skillin the art that an individual on whom any method of the invention ispracticed does not have, e.g., congestive heart failure, chronic heartfailure, hypertension, diabetic neuropathy, migraine, a predispositionto myocardial infarction, or any other condition for which ARBs areindicated, or is not otherwise in need of treatment. An individual whohas a condition or disease described herein can receive an ARB inconjunction with an antigen for the purpose of enhancing an immuneresponse if the individual is not being treated with an ARB.

In certain embodiments, a compound of Formula (I) (e.g., Ondansetron orAlosetron), or a combination of compounds of Formula (I) can beadministered in conjunction with the antigen, vaccine, or anti-tumorpreparation. In certain embodiments, a compound or combination ofcompounds of Formula (I) (e.g., Ondansetron and/or Alosetron) can beadministered in conjugation with an antigen, vaccine, or anti-tumorpreparation to individuals who are otherwise not receiving Ondansetronand/or Alosetron for treatment of a pre-existing condition. As notedelsewhere herein, Ondansetron and/or Alosetron can be prescribed for thetreatment and/or prevention of a variety of diseases. Accordingly, itwill be appreciated by one of skill in the art that an individual onwhom any method of the invention is practiced does not have, e.g.,irritable bowel syndrome (IBS), post-operative nausea and vomiting(PONV), radiation-induced nausea and vomiting (RINV),chemotherapy-induced nausea and vomiting (CINV), or is not otherwise inneed of Ondansetron treatment and/or Alosetron treatment. An individualwho has a condition or disease described herein, or is otherwise in needof treatment, can receive Ondansetron, Alosetron in conjunction with anantigen, vaccine, or anti-tumor preparation for the purpose of enhancingan immune response if the individual is not being treated withOndansetron and/or Alosetron.

Alternatively, in some embodiments, the individuals who are beingtreated with Ondansetron and/or Alosetron can temporarily suspendedOndansetron and/or Alosetron treatment. For example, an individual whois being treated with Ondansetron and/or Alosetron can receiveOndansetron, Alosetron, and/or a combination of compounds of Formula (I)comprising Ondansetron and/or Alosetron in conjunction with an antigen,vaccine, or anti-tumor preparation if the individual has takenOndansetron and/or Alosetron more than about 1 hour, more than about 3hours, more than about 6 hours, more than about 12 hours, more thanabout 18 hours, more than 24 hours, more than 1 day more than 2 days,more than 3 days, more than 4 days, more than 5 days, more than 6 days,more than 1 week, more than 3 weeks, more than 1 month, more than threemonths, more than 6 months, or more than 1 year before receivingOndansetron, Alosetron, and/or a combination of compounds of Formula (I)comprising Ondansetron and/or Alosetron in conjunction with the antigen,including any range in between these values. Individuals receivingOndansetron and/or Alosetron therapy can temporarily suspend treatmentprior to being administered Ondansetron, Alosetron, and/or a combinationcompounds of Formula (I) comprising Ondansetron and/or Alosetron inconjunction with an antigen, vaccine, or anti-tumor preparation for thepurpose of enhancing an immune response, inhibiting monocyte migrationto a lymph node, amplifying vaccine immunity, or treating cancer. Forexample, blood and/or urine samples provided by such an individual canbe assayed for the presence of Ondansetron and/or Alosetron (and/or itsmetabolites) using chromatographic or spectroscopic methods describedelsewhere herein. The methods of the invention can be practiced on theindividual once it has been determined that Ondansetron and/or Alosetron(and/or its metabolites) is undetectable in the individual's bloodand/or urine. Such methods are described elsewhere herein.

Similarly, in some embodiments, the individuals who are being treatedwith an ARB can temporarily suspended ARB treatment. For example, anindividual who is being treated with an ARB for a pre-existing conditioncan receive an ARB in conjunction with an antigen, vaccine, oranti-tumor preparation if the individual has taken the ARB more thanabout 1 hour, more than about 3 hours, more than about 6 hours, morethan about 12 hours, more than about 18 hours, more than 24 hours, morethan 1 day more than 2 days, more than 3 days, more than 4 days, morethan 5 days, more than 6 days, more than 1 week, more than 3 weeks, morethan 1 month, more than three months, more than 6 months, or more than 1year before receiving the ARB in conjunction with the antigen, includingany range in between these values. Alternatively, in some embodiments,an individual who is being treated with an ARB for a pre-existingcondition can suspend ARB treatment and be tested, using methods wellknown in the art, to determine that the individual does not have adetectable level of an ARB in their blood and/or urine prior to theadministration of the ARB in conjunction with the antigen, the vaccine,or the anti-tumor preparation. For example, blood and/or urine samplesprovided by such an individual can be assayed for the presence of an ARB(and/or its metabolites) using chromatographic or spectroscopic methodsdescribed elsewhere herein. The methods of the invention can bepracticed on the individual once it has been determined that the ARB(and/or its metabolites) is undetectable in the individual's bloodand/or urine. Such methods are described elsewhere herein.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and scope of the appended claims.

Example 1 Losartan Blocked Canine and Human Monocyte Migration In Vitro

Boyden Chamber Assay.

The Boyden chamber assay is based on a chamber of two medium-filledcompartments separated by a microporous membrane. In general, cells areplaced in the upper compartment and are allowed to migrate through thepores of the membrane into the lower compartment, in which chemotacticagents are present. After an appropriate incubation time, the membranebetween the two compartments is fixed and stained, and the number ofcells that have migrated to the lower side of the membrane isdetermined. Experiments using this assay were performed as described inMitchell et al. (2012) Int. Immunopharmacol. 15: 357-363 to assess themigration of monocytes.

A Boyden chamber assay was performed to assess the migration of dogmonocytes and of human monocytes (i.e., the human monocyte cell lineTHP-1) in response to a CCL2 (MCP-1) gradient and to assess the abilityof Losartan to inhibit monocyte migration in vitro. As shown in FIG. 1,Losartan potently blocked human monocyte migration (THP-1) in vitro in adose dependent manner at concentrations easily achievable in vivo(hpf=high power field). The asterisk in FIG. 1 indicates statisticaldifferences, i.e., a p-value<0.05. FIG. 2 shows that Losartan potentlyblocked canine monocyte migration in vitro in a dose dependent manner atconcentrations easily achievable in vivo. The asterisks in FIG. 2indicate statistical differences, i.e., a p-value<0.05. Moreover, atconcentrations of 1 μg/ml and 10 μg/ml, Losartan blocked canine monocytemigration even in the presence of MCP-1. Taken together, these resultsindicate that Losartan is an inhibitor of monocyte migration in vitro.

Example 2 Ondansetron Blocked Human Monocyte Migration In Vitro

A Boyden chamber assay was performed (as described in Mitchell et al.(2012) Int. Immunopharmacol, supra) to assess the migration of humanmonocytes (i.e., the human monocyte cell line THP-1) in response to aCCL2 (MCP-1) gradient and to assess the ability of Ondansetron toinhibit monocyte migration in vitro. As shown in FIG. 3, Ondansetronpotently blocked THP-1 monocyte migration in vitro in a dose dependentmanner at concentrations easily achievable in vivo. The single asteriskin FIG. 3 indicates statistical differences, i.e., a p-value<0.05. Thedouble asterisks in FIG. 3 indicates statistical differences, i.e., ap-value<0.01. These results indicate that Ondansetron is an inhibitor ofmonocyte migration in vitro.

Example 3 Ondansetron Treatment Inhibited Monocyte Migration to LymphNodes in Mice

Footpad inflammation-induced monocyte migration assays were performed aspreviously described (Mitchell et al. (2012), J. Immunology 189:5612-5621) to determine whether Ondansetron inhibits monocyte migrationin vivo in mice. Briefly, two groups of mice (n=4 per group) wereinjected with 50 μl of liposomal vaccine adjuvant in one footpad. Thefirst group of mice was treated by injection of 3 mg/kg Ondansetronadministered IP at the time of footpad injection and the Ondansetroninjections were repeated 12, 24, and 36 hours following footpadinjection. The second group of mice did not receive any additionaltreatment. The third group received neither the vaccine nor theOndansetron. Inflammatory monocyte migration to the draining popliteallymph nodes was assessed in all three groups of mice via flow cytometry(as described in Mitchell et al. (2012), J. Immunology, supra) 24 hoursfollowing vaccination.

As shown in FIG. 4, the migration of inflammatory monocytes to thedraining lymph nodes was significantly reduced in mice who receivedOndansetron in conjunction with the vaccine as compared to mice whoreceived no additional treatment with vaccination. FIG. 5 provides aquantitative analysis of the results depicted in FIG. 4. The tripleasterisks in FIG. 5 indicates statistical differences, i.e., ap-value<0.001. These results indicate that Ondansetron acts as a potentinhibitor of inflammatory monocyte migration in vivo.

Example 4 Losartan Treatment Inhibited Monocyte Migration to Lymph Nodesin Mice

Footpad inflammation-induced monocyte migration assays were performed asdescribed previously (as described in Mitchell et al. (2012), J.Immunology, supra) to determine whether Losartan inhibits monocytemigration in vivo in mice. Briefly, two groups of mice (n=4 per group)were vaccinated in the right rear footpad. The first group of mice wastreated by injection of 30 mg/kg Losartan at the time of injection and12 hours following injection. The second group of mice did not receiveany additional treatment. Inflammatory monocyte migration to thedraining popliteal lymph nodes was assessed in all three groups of micevia flow cytometry (as described in Mitchell et al. (2012), J.Immunology, supra) 24 hours following vaccination.

As shown in FIG. 6, the migration of inflammatory monocytes to thedraining lymph nodes was significantly reduced in mice who receivedLosartan in conjunction with the vaccine as compared to mice whoreceived no additional treatment with vaccination. The asterisk in FIG.6 indicates statistical differences, i.e., a p-value<0.05. These resultsindicate that Losartan acts as a potent inhibitor of inflammatorymonocyte migration in vivo.

Example 5 Losartan Treatment Inhibited Monocyte Migration to Lymph Nodesin Dogs

To determine whether Losartan inhibits monocyte migration in vivo, dogs[n=2] were treated with Losartan at 2 mg/kg/day for 48 hours. Bloodsamples were then taken from the dogs, and Boyden chamber assays wereperformed as described previously (Mitchell et al. (2012) Int.Immunopharmacol, supra) As shown in FIG. 7(a and b), monocyte migrationwas significantly inhibited in blood samples obtained from the dogsafter Losartan treatment (FIG. 7 a shows dog 1, FIG. 7b shows dog 2; theleft panel is a no migration control, the middle panel shows monocytemigration prior to losartan treatment, and the right panel showsmonocyte migration after 48 hours of Losartan treatment). The tripleasterisks in FIGS. 7a and 7b indicate statistically significantdifferences (p<0.05). This experiment was repeated in 3 dogs withcancer. Briefly, each of the three dogs was treated for two weeks with 2mg/kg/day Losartan before the Boyden chamber assay was performed. FIG. 8provides the results of pooled monocyte migration data (the left panelis a no migration control, the middle panel shows monocyte migrationprior to Losartan treatment, and the right panel shows monocytemigration after 2 weeks of Losartan treatment). Monocyte migration wasinhibited in blood samples obtained from the 3 dogs with cancer who hadbeen receiving Losartan treatment for two weeks. Taken together, theseresults indicate that Losartan treatment at a concentration of 2mg/kg/day for at least 48 hours can inhibit monocyte migration in vivo.

Example 6 Losartan Treatment at Time of Vaccination SignificantlyImproved Humoral and Cellular Immunity

To assess the effects of Losartan on humoral immunity, mice were treatedwith Losartan at the time of vaccination. Briefly, two groups of mice(n=5) were vaccinated with 5 μg of ovalbumin. The first group of micereceived 30 mg/kg Losartan via injection at the time of vaccination, andat 12, 24, and 36 hours following vaccination. The second group of micereceived no additional treatment following vaccination. Secondvaccinations were repeated 10 days following the initial vaccinations.Second Losartan treatments were repeated for the first group of mice asdescribed. Anti-ovalbumin antibodies from each group of mice weretitered (as described in Mitchell et al. (2012), J. Immunology, supra)to assess the degree to which humoral immunity was enhanced in the micereceiving Losartan treatment.

As shown in FIG. 9, mice treated with Losartan were found to havesignificantly higher anti-ovalbumin antibody titers than mice whoreceived no additional treatment with vaccination, indicating thatadministration of Losartan in conjunction with the antigen ovalbuminenhanced humoral immunity. The asterisk in FIG. 9 indicates statisticaldifferences, i.e., a p-value<0.05.

Example 7 Ondansetron Treatment at Time of Vaccination SignificantlyImproved Humoral Immunity

To assess the effects of Ondansetron on humoral immunity, mice weretreated with Ondansetron at the time of vaccination. Briefly, two groupsof mice (n=4 per group) were vaccinated subcutaneously with 1 μg ofovalbumin in a liposomal adjuvant. The first group of mice received 3mg/kg Ondansetron via i.p. injection at the time of vaccination, and at12, 24, and 36 hours following vaccination. The second group of micereceived no additional treatment following vaccination. A third group ofmice received neither the vaccine nor the Ondansetron. Secondvaccinations were repeated for the first two groups of mice 10 daysfollowing the initial vaccinations, and second Ondansetron treatmentswere repeated for the groups as described for the first cycle ofvaccination. Anti-ovalbumin antibodies from each group of mice weretitered (as described in Mitchell et al. (2012), J. Immunology, supra)to assess the degree to which humoral immunity was enhanced in the micereceiving Ondansetron treatment.

As shown in FIG. 10, mice treated with Ondansetron were found to havesignificantly higher anti-ovalbumin antibody titers than mice whoreceived no additional treatment with vaccination, indicating thatadministration of Ondansetron in conjunction with the antigen ovalbuminenhanced humoral immunity. The asterisk in FIG. 10 indicates indicatestatistical differences, i.e., a p-value<0.05.

Example 8 Losartan Amplifies Vaccine Cellular Immunity

Two groups of mice were immunized as noted in Example 7 above. Todetermine whether cellular immunity was enhanced in the Losartan-treatedmice, mice from both the first and second groups were euthanized, andtheir spleen cells were assayed to determine IFNγ secretion levels inresponse to ovalbumin restimulation. Splenectomies and restimulationassays for cellular immune responses were performed as previouslydescribed (Mitchell et al. (2012), J. Immunology, supra). Spleen cellswere restimulated in vitro for 72 hours with 5 μg/ml ovalbumin protein.

As shown in FIG. 11, spleen cells from mice that were vaccinated andtreated concurrently with Losartan produced significantly more IFNγ,indicating a significant increase in T cell responses to vaccinationwhen losartan was administered in conjunction with the antigenovalbumin. The asterisks in FIG. 11 indicate statistical differences,i.e., a p-value<0.05.

Example 9 Ondansetron Administration Enhanced Tumor Vaccine Responses

The A20 mouse B-cell lymphoma model was used to assess the effects ofOndansetron on tumor vaccine responses. Briefly, two groups of mice (n=5per group) with established d3 A20-HA tumors (i.e., tumors expressingHA) were vaccinated weekly for 3 weeks with HA antigen (1 μg per mousein liposome adjuvant), either without or with concurrently administeredondansetron (3 mg/kg i.p., administered 4 times at 12 h intervals). Athird group of mice received neither the HA nor the Ondansetron. Tumormeasurements were made using calipers every 2-3 days. Mice weresacrificed when their tumors reached a diameter of 1.5 cm.

As shown in FIG. 12, tumor growth rates were significantly reduced inthe mice that received the vaccine in conjunction with the Ondansetron,as compared to tumor growth rates in mice that received only thevaccine. The triple asterisks in FIG. 12 indicate statisticaldifferences, i.e., a p-value<0.001. Single asterisks indicate that thereare statistically significant differences in tumor sizes between thegroup of mice who had received Ondansetron and the vaccine and the groupof mice that received only the vaccine.

FIG. 13 shows tumor tissues from a mouse that received only the vaccine(top panel) and tumor tissues from a mouse that received vaccine inconjunction with Ondansetron (bottom panel) that were evaluated viaimmunohistochemistry. Labeled anti-CD11b antibodies were used toquantitate the numbers of infiltrating CD11b+ monocyte/macrophages inthe tumor tissues. Tumors of mice treated with ondansetron had markedlyfewer monocyte/macrophages than tumors of mice that received the vaccinealone. Taken together, the results provided in FIGS. 12 and 13demonstrate that administering Ondansetron in conjunction with ananti-tumor vaccine reduced inflammatory monocyte migration into tumortissues and reduced tumor growth.

Example 10 Synergistic Combination of Losartan and Sunitinib

To assess the effect of a Losartan/Sunitinib combination therapyapproach for tumor treatment, tumors were seeded in mice (BALB/c, n=5per group) via intravenous injection with 2.5×10⁵ K7M2 osteosarcomacells expressing a luciferase reporter gene. Three days later, treatmentwas started with losartan (6 mg/kg, i.p. BID) and sunitinib (0.8 mg/kg,ip, once daily for 5 days on, 2 off). Drugs were injected separately.Tumor growth was monitored by IVIS imaging every 2-3 days.

As shown in FIG. 14, tumors in mice receiving the Losartan/Sunitinibcombination, had a decreased rate of growth as compared to micereceiving Losartan or Sunitinib alone (data not significant), suggestingthat these drugs may interact synergistically to suppress tumor growthand metastasis. At the completion of the experiment, all animals wereeuthanized and lung tissues collected to evaluate myeloid cell responsesby flow cytometry. FIG. 15(a) shows the percentage of CD11b+/Ly6C+monocytes, as compared to total monocytes present in the lungs of thesemice, and FIG. 15(b) shows the percentage of CD11b+/Ly6C+/PDL1+monocytes compared to the total cells present in the lungs of thesemice. Using each of these metrics, a trend was observed suggesting thatLosartan/Sunitinib combination results in a synergistic inhibition ofmonocyte migration to the lung tissue having tumors.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, PCT patent application, PCT patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification or listed inany Application Data Sheet are incorporated herein by reference in theirentirety. From the foregoing it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit and scope of the invention.

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What is claimed is:
 1. A method of amplifying vaccine immunity in anindividual, the method comprising administering to the individual thevaccine in conjunction with an effective amount of a compound of Formula(I):

wherein R¹ is hydrogen or C₁₋₆alkyl;

is a single bond or double bond; Q¹ is N or CH; R² is selected fromhydrogen and C₁₋₆ alkylene, wherein one carbon unit of said alkylene isoptionally replaced with —O—, —S—, —SO—, —SO₂—, —NR^(a)—, or —CO—;wherein R^(a) is hydrogen or C₁₋₆ alkyl; and R³ is hydrogen or anoptionally substituted 5-membered heteroaryl ring; wherein theheteroaryl ring is optionally substituted with C₁₋₆alkyl; orpharmaceutically acceptable salts thereof.
 2. The method of claim 1,wherein the vaccine comprises live whole virus, killed whole virus,attenuated whole virus, killed bacteria, attenuated bacteria, avirus-like particle, a bacterial, viral, or parasite protein, arecombinant protein, or a peptide.
 3. The method of claim 1, wherein thecompound of Formula (I) and the vaccine are present in a singlepharmaceutical composition.
 4. The method of claim 1, wherein thecompound of Formula (I) is present in a first pharmaceutical compositionand wherein the vaccine is present in a second pharmaceuticalcomposition.
 5. The method of claim 1, wherein the compound of Formula(I) is a compound of Formula (II):

wherein R¹ is hydrogen or C₁₋₆ alkyl;

is a single bond or double bond; Q¹ is N or CH; and R⁴ is an optionallysubstituted 5-membered heteroaryl ring; wherein the heteroaryl ring isoptionally substituted with C₁₋₆alkyl; or pharmaceutically acceptablesalts thereof.
 6. The method of claim 1, wherein the compound of Formula(I) is a compound of Formula (III):

wherein

is a single bond or double bond; Q¹ is N or CH; and R⁴ is an optionallysubstituted 5-membered heteroaryl ring; wherein the heteroaryl ring isoptionally substituted with C₁₋₆alkyl; or pharmaceutically acceptablesalts thereof.
 7. The method of claim 1, wherein the compound of Formula(I) is a compound of Formula (IV):

wherein R¹ is hydrogen or C₁₋₆ alkyl;

is a single bond or double bond; Q¹ is N or CH; and R⁴ is selected from

or pharmaceutically acceptable salts thereof.
 8. The method of claim 1,wherein the compound of Formula (I) is a compound of Formula (V):

wherein

is a single bond or double bond; Q¹ is N or CH; and

R⁴ is selected from or pharmaceutically acceptable salts thereof.
 9. Themethod of claim 1, wherein the compound of Formula (I) is Ondansetron.10. The method of claim 9, wherein the Ondansetron is administered at adosage of less than 12 mg.
 11. The method of claim 9, wherein theindividual has not taken Ondansetron for the treatment of irritablebowel syndrome (IBS), post-operative nausea and vomiting (PONY),radiation-induced nausea and vomiting (RINV), or chemotherapy-inducednausea and vomiting (CINV).
 12. The method of claim 9, wherein theindividual does not have a detectable level of Ondansetron in theirblood or urine prior to administration of the Ondansetron in conjunctionwith the vaccine.
 13. The method of claim 1, wherein the compound ofFormula (I) is Alosetron.
 14. The method of claim 13, wherein theAlosetron is administered at a dosage of less than 0.5 mg.
 15. Themethod of claim 13, wherein the individual has not taken Alosetron forthe treatment of irritable bowel syndrome (IBS), post-operative nauseaand vomiting (PONY), radiation-induced nausea and vomiting (RINV), orchemotherapy-induced nausea and vomiting (CINV).
 16. The method of claim13, wherein the individual does not have a detectable level of Alosetronin their blood or urine prior to administration of the Alosetron inconjunction with the vaccine.